General detection and isolation of specific cells by binding of labeled molecules

ABSTRACT

The present invention relates to detection molecules comprising at least one binding molecule, at least one linker and at least one label, and detection methods making use of same. The invention provides a high-throughput method for detection, isolation and/or identification of specific entities or cells.

The application claims priority from PA 2014 00311 filed Jun. 13, 2014,PA 2014 00322 filed Jun. 18, 2014 and PA 2014 00453 filed Aug. 16, 2014.All patent and non-patent references cited in the present application,are hereby incorporated by reference in their entirety.

FIELD OF INVENTION

The present invention relates to detection molecules comprising at leastone binding molecule, at least one linker and at least one label, anddetection methods making use of same. The invention provides ahigh-throughput method for detection, isolation and/or identification ofspecific entities or cells.

BACKGROUND OF INVENTION

The adaptive immune system is directed through specific interactionsbetween immune cells and antigen-presenting cells (e.g. dendritic cells,B-cells, monocytes and macrophages) or target cells (e.g. virus infectedcells, bacteria infected cells or cancer cells). In important field inimmunology relates to the understanding of the molecular interactionbetween an immune cell and the target cell.

Specifically for T-lymphocytes (T-cells), this interaction is mediatedthrough binding between the T-cell receptor (TCR) and the MajorHistocompatibility Complex (MHC) class I or class II. The MHC moleculescarries a peptide cargo, and this peptide in decisive for T-cellrecognition. The understanding of T-cell recognition experienced adramatic technological breakthrough when Atman et al. (1) in 1996discovered that multimerization of single peptide-MHC molecules intotetramers would allow sufficient binding-strength (avidity) between thepeptide-MHC molecules and the TCR to determine this interaction througha fluorescence label attached to the MHC-multimer. Suchfluorescent-labelled MHC multimers (of both class I and class IImolecules) are now widely used for determining the T-cell specificity.The MHC multimer associated fluorescence can be determined by e.g. flowcytometry or microscopy, or T-cells can be selected based on thisfluorescence label through e.g. flow cytometry or bead-based sorting.However, a limitation to this approach relates to the number ofdifferent fluorescence labels available, as each fluorescence labelserve as a specific signature for the peptide-MHC in question.

Thus, this strategy is poorly matching the enormous diversity in T-cellrecognition. For the most predominant subset of T-cells (the αβ TCRT-cells), the number of possible distinct αβ TCRs has been estimated at˜1015 (2) although the number of distinct TCRs in an individual human isprobably closer to 107(3). Therefore, much effort has attempted toexpand the complexity of the T-cell determination, with the aim toenable detection of multiple different T-cell specificities in a singlesample. A more recent invention relates to multiplex detection ofantigen specific T-cells is the use of combinatorial encoded MHCmultimers. This technique uses a combinatorial fluorescence labellingapproach that allows for the detection of 28 different T-cellpopulations in a single sample when first published (4,5), but has laterbeen extended through combination with novel instrumentation and heavymetal labels to allow detection of around 100 different T-cellpopulations in a single sample (6).

The requirement for new of technologies that allow a more comprehensiveanalysis of antigen-specific T-cell responses is underscored by the factthat several groups have tried to develop so-called MHC microarrays. Inthese systems, T-cell specificity is not encoded by fluorochromes, butis spatially encoded (7,8). In spite of their promise, MHC microarrayshave not become widely adopted, and no documented examples for its valuein the multiplexed measurement of T-cell responses, for instance epitopeidentification, are available.

Considering the above, there remains a need for a high-throughput methodin the art of detection, isolation and/or identification of specificantigen responsive cells, such as antigen specific T-cells.

Further, there remains a need for a high-throughput method in the art ofdetection, isolation and/or identification of specific entities orcells, which cells can be detected via a binding molecule specific forsaid entity or cell.

Further, there remains a need in the art, considering the often limitedamounts of sample available, for methods allowing detection, isolationand/or identification of multiple species of specific entities or cells,such as specific antigen responsive cells, such as T-cells, in a singlesample.

SUMMARY OF INVENTION

The present invention provides a detection molecule comprising a bindingmolecule (BM), a linker (Li) and a label (La). Said detection moleculecan be used in various methods, such as methods comprising one or moresteps of recognizing at least one entity or cell, binding at least oneentity or cell, detecting at least one entity/cell-detection moleculecomplex, isolating at least one entity/cell-detection molecule complexand/or identifying one or more detection molecules bound to at least oneentity/cell.

It is an aspect of the invention to provide a detection methodcomprising the steps of

-   -   1. Combining a sample with at least one detection molecule;        wherein the detection molecule comprises a binding molecule        (BM), a linker (Li), and a label (La); and wherein said sample        comprises at least one cell and/or entity,    -   2. Incubating the at least one detection molecule and the        sample;    -   3. Isolating and/or detecting the at least one detection        molecule of step b), and    -   4. Optionally determining the identity of the at least one        detection molecule of step c).

A binding molecule is a molecule that specifically associates covalentlyor non-covalently with a structure belonging to or associated with anentity in a sample. The defined structure in sample bound by a bindingmolecule is also called the target structure or target of the bindingmolecule.

The label comprised in the detection molecule of the present inventionis any molecule, atom or signal the identity of which can be found ordetermined by any means. The label is unique and specific for a certaindetection molecule, or set of detection molecules, and enables theisolation, detection and/or identification of the identity of saiddetection molecules (or cell-detection molecule complexes). In aparticular embodiment the label is a nucleic acid label, such as a DNAlabel,

The linker comprised in the detection molecule of the present inventionis a molecular entity and/or bond that connect the binding molecule andthe label of the detection molecule.

The present invention describes the use of labels, preferably nucleicacid labels (comprising a barcode sequence) as specific labels forbinding molecules such as MHC multimers to determine e.g. the antigenresponsiveness in biological samples. After cellular selection thebarcode sequence of the label can be revealed by e.g. sequencing. Thistechnology allows for detection of multiple (potentially >1000)different antigen-specific cells in a single sample.

DESCRIPTION OF DRAWINGS

FIG. 1 . Structure of detection molecules. (A) Structure of a detectionmolecule, in which the “binding molecule” comprises n pMHC complexes,where n can be any integer between 1 and 1000. (B) Structure of adetection molecule, comprising “binding molecule”, label and the linkerconnecting the binding molecule and the label. The lower half of thefigure shows the symbols representing the DNA tag (also called theoligonucleotide or oligonucleotide tag), the linker connecting bindingmolecule and label, MHC (major histocompatibility complex), peptide orother molecule (that binds to the MHC complex), pMHC (peptide-majorhistocompatibility complex), and the binding molecule (BM).

FIG. 2 . Isolation and detection of detection molecules capable ofbinding to cells. The different steps of the method is outlined. In thedepicted method the label of the detection molecule is anoligonucleotide. In Step 1 cells and “molecule library” (2-1015detection molecules) is mixed. In Step 2 the cells and detectionmolecules are incubated, allowing none, some or all of the detectionmolecules to bind cells, and vice versa, allowing none, some or all ofthe cells to bind detection molecules. In step 3 the cell-detectionmolecule complexes are isolated, enriched for or detected. In thedepicted approach, cells are spun down, having the effect of spinningdetection molecules bound to the cells down as well. In Step 4 thedetection molecules that bound to cells in step 3 (and therefore wereco-precipitated with the cells) are identified. In the example the labelconsists of an oligonucleotide. Therefore, by PCR amplification of theoligonucleotide labels, followed by e.g. polyacrylamide gelelectrophoresis to distinguish labels with different migration in thegel, the labels may be identified and thereby the detection molecules(and the identity of the corresponding binding molecules) can beidentified. Alternatively, the oligonucleotide labels are identified bysequencing, directly from the precipitate of step 3, or followingamplification of the oligonucleotides in the precipitate by e.g. PCR.

FIG. 3 . Generation of barcode-labeled MHC multimers.

FIG. 3 describes how peptide-MHC molecules, nucleic acid (DNA)-barcodesand (optional) fluorescent labels are assembled to form a library of MHCmultimers each holding a DNA-barcode specific for the given peptide-MHCmolecule involved. FIG. 3A: The barcode is designed to have a uniquesequence that can be determined through DNA sequencing. Also the barcodehave shared amplification ends, enabling amplification of allDNA-barcodes simultaneously in a PCR reaction. DNA-barcodes are attachedto the MHC-multimerization backbone (e.g. via a biotin linker binding tostreptavidin on the multimer backbone). FIG. 3B: Represents the multimerbackbone. This may be any backbone that allow multimerization ofmacro-molecules. The backbone may (optionally) hold a fluorescence label(illustrated by the asterisk) to track the total pool of MHC multimerbinding cells irrespectively of the peptide-MHC multimer specificity.FIG. 3C: Represents the peptide-MHC molecule of interest, carrying aspecific peptide cargo (horizontal line). FIG. 3D: Represents theassembled peptide-MHC mulitimers carrying the DNA barcode.

FIG. 4 . Generation of a library of barcode labelled MHC multimers(Composition).

FIG. 4 illustrates the generation of a full barcode library. FIG. 4A:This library is composed of multiple, potentially more than 1000different peptide-MHC multimers, each with a specific DNA-barcode. Suchthat barcode #1 codes for peptide-MHC complex #1, barcode #2 codes forpeptide-MHC complex #2, barcode #3 codes for peptide-MHC complex #3, andso on until the possible mixture of thousands different specificitieseach with a specific barcode. FIG. 4B: Represents the final reagent,which is a mixture of numerous different MHC-multimers each carrying aspecific DNA barcode as a label for each peptide-MHC specificity.

FIG. 5 . Detection of antigen responsive cells in a single sample.

Method for detecting antigen responsive cells in a sample. In FIG. 5 itis illustrated how this library can be used for staining of antigenresponsive cells in a single sample. FIG. 5A: Cells in single cellsuspension (may e.g., but not exclusive, originate from peripheralblood, tissue biopsies or other body fluids) are mixed with thepeptide-library represented in FIG. 4B. FIG. 5B: After staining, cellsare sequentially washed and spun to remove residual MHC multimers thatare not bound to a cellular surface. Specific cell populations, e.g.T-cells (CD8 or CD4 restricted), other immune cells or specifically MHCmultimer binding T-cells may be sorted by flow cytometry or others meansof cell sorting/selection. FIG. 5C: The DNA-barcode oligonucleotidesequences isolated from the cell population is amplified by PCR. FIG.5D: This amplification product is sequenced by deep sequencing(providing 10-100.000s of reads). The sequencing will reveal thespecific barcode sequence of DNA barcodes attached to cells in thespecimen after selection, as these will appear more frequent thansequences associated to the background of non-specific attachment of MHCmultimers. The “signal-to-noise” is counteracted by the fact that anyunspecific MHC multimer event will have a random association of 1/1000different barcodes (dependent of the size of the library), making iteven more sensitive than normal multimer staining.

FIG. 6 . Describes the possibility of linking the antigen specificity(tracked by the barcode) to other properties. Use of a multimeric majorhistocompatibility complex. In FIG. 6 , it is illustrated how thistechnology can be used to link different properties to the antigenspecificity of a cell population.

FIG. 6A: Illustrates how cells after binding to a barcode labeled MHCmultimer library may be exposed to a certain stimuli. Cell populationscan be selected based on the functional response to this stimuli (e.g.,but not exclusive, cytokine secretion, phosphorylation, calcium releaseor numerous other measures). After selecting the responsive ornon-responsive population (following the steps of FIG. 5 ), the DNAbarcodes can be sequenced to decode the antigen responsiveness, andthereby determining the antigen-specificities involved in a givenresponse.

FIG. 6B: Illustrates how cells can be selected based on phenotype, tolink a certain set of phenotypic characteristics to theantigen-responsiveness.

FIG. 6C: Represents the possibility for single-cell sorting ofMHC-multimer binding cells based on the co-attached fluorescence labelon the MHC multimer. Through single-cell sorting the antigen-specificityof the given cell can be determined on a single cell level throughsequencing of the associated barcode label. This can be linked to theTCR that can also be sequenced on a single cell level, as recentlydescribed (10). Hereby, this invention will provide a link between theTCR sequence, or other single-cell properties and the antigenspecificity, and may through the use of barcode labeled MHC multimerlibraries enable definition of antigen-specific TCRs in a mixture ofthousands different specificities.

FIG. 6D illustrates the use of barcode labeled MHC multimer librariesfor the quantitative assessment of MHC multimer binding to a givenT-cell clone or TCR transduced/transfected cells. Since sequencing ofthe barcode label allow several different labels to be determinedsimultaneously on the same cell population, this strategy can be used todetermine the avidity of a given TCR relative to a library of relatedpeptide-MHC multimers. The relative contribution of the differentDNA-barcode sequences in the final readout is determined based on thequantitative contribution of the TCR binding for each of the differentpeptide-MHC multimers in the library. Via titration based analyses it ispossible to determine the quantitative binding properties of a TCR inrelation to a large library of peptide-MHC multimers. All merged into asingle sample. For this particular purpose the MHC multimer library mayspecifically hold related peptide sequences or alanine-substitutionpeptide libraries.

FIG. 7 . Experimental data for attaching a DNA-barcode to a MHC multimerand amplify the specific sequences following T-cell staining. FIG. 7A:Shows the staining of cytomegalovirus (CMV) specific T-cells in aperipheral blood samples. The specific CMV-derived peptide-MHC multimerswas labeled with a barcode (barcode #1) and mixed with anirrelevant/non-specific peptide-MHC multimer (HIV) labeled with barcode(barcode #2) and mixed with 998 other non-barcode labeled non-specificMHC multimers. Data here shows the feasibility for staining ofCMV-specific T-cells in a mixture of 1000 other MHC multimers. Data isshown for three different staining protocols (A, B, C). FIG. 7B: Showsthe readout of the specific barcode sequences by quantative PCR. Barcode#1 (CMV) determining the CMV specific T-cell in detected for all threestaining protocols, whereas the irrelevant/non-specific barcode signal,barcode #2 (HIV) is undetectable.

FIG. 8 : Stability of single-stranded and double-strandedoligonucleotides in blood preparations, cf. Example A.

FIG. 9 : A schematic presentation of the Label systems applied; the 1OSand 2OS DNA-barcode systems. A. The 1OS system comprised of singlestranded oligonucleotides which were applied as a DNA barcodes. 1OSstructure: A ˜80 nucleotide sequence. Biotin is attached at the 5′endwhere it accommodates easy attachment via a streptavidin binding site.The nucleotide sequence consists of a 20-25 nt forward primer followedby a random incorporation of 6 nt (N6), a short 3 nt linker and 25 ntthat comprise the diverse barcode-identity. This is followed by 20-25 ntreverse primer region. B. The 2OS system are generated from annealingand elongation of two partially complementaryoligonucleotides-nucleotide sequences, Oligo A and Oligo B, producing afully double-stranded unique DNA sequences, which are used asDNA-barcodes. Oligo A corresponds largely to the 1OS DNA-barcodes, i.e.biotin is coupled to the 5′-end which begins with a forward primerregion that is followed by a N6 segment and the uniqueoligonucleotide-sequence that will constitute one half of the barcodeidentity. After the unique sequence the reverse primer region of the 1OSsystem is replaced by a 16 nt sequence that is complementary to the3′-end of Oligo B. Oligo B is described from the 3′-end where it annealswith Oligo A with a 16 nt complementary binding region followed by theunique oligonucleotide-sequence that will constitute the other half ofthe barcode identity. Hereafter comes a N6 region, which is followed bywhat is actually a forward primer region (since it lies in the 5′-end)but its complementary region will constitute the reverse priming regionof the 2OS-DNA barcodes. Thus the primary differences between 1OS and2OS barcodes are revealed in a greater length of the 2OS barcode, whichis required to encompass two unique barcode regions, an annealing-siteand an additional N6 region nearest the 3′-end. This adds up to ˜130nts. C. Through PCR amplification of enriched DNA-barcode Labels theseare appointed a sample identification barcode (6-8 nt), which is part ofthe forward primer design. Additionally both the forward and reverseprimers hold adaptors for the Ion Torrent sequencing (A-key and P1-keyrespectively). Primers and keys for the 1OS and the 2OS barcode were ofa similar design (here showing a 2OS barcode). Asterisk=biotin,nt=nucleotide.

FIG. 10 : Detection of a CMV specificity amongst negative controlDetection Molecules (cf Example 1). A unique 2OS DNA-barcode wasassociated with the CMV positive Detection Molecules in 1., whileanother unique 2OS DNA-barcode was associated with the CMV positivecontrol Detection Molecules in 2. The spare DNA-barcode in each sampleincubation was associated with the HIV negative control DetectionMolecule. A. Representative dot plot showing the PE positive populationafter staining with CMV and HIV Detection Molecules carrying separate2OS DNA-barcodes. B. Ct values from multiplex qPCR of the sortedPE-pMHC-dextramer positive cells. Cells were stained with 1. and 2.respectively. Detection Molecules associated with a positive control(CMV) 2OS barcode and a negative control (HIV) 2OS barcode were presentduring staining, but the negative control (HIV) Detection Molecule wasevidently washed out. The results obtained from two individualexperiments are presented in separate bars. Approximately 200 cells wereapplied in each PCR. QPCR was run in duplicates and Ct values are shownas mean±range of duplicates.

FIG. 11 : Detection of a CMV specificity amongst negative controlDetection Molecules (cf. Example 2). A unique 1OS DNA-barcode wasassociated with the positive control Detection Molecules in 1, whileanother unique barcode was associated with the positive control reagentsin 2. The spare barcode in each experiment was associated with a HIVnegative Detection Molecules. In addition 998× unlabeled (i.e. lacking aDNA-barcode) negative control Detection Molecules were present inboth 1. and 2. A, Ct values from multiplex qPCR of the sortedPE-pMHC-dextramer positive cells. Cells were stained with 1. and 2.respectively. Detection Molecules associated with a positive control(CMV) 1OS barcode and a negative control (HIV) 1OS barcode were presentduring staining, but the negative control (HIV) Detection Molecules wereevidently washed out. Approximately 575 cells were analyzed in eachqPCR. B. The estimated number of barcodes bound per cell relative to theobtained Ct-values (as calculated from a standard curve of 1OS barcode).It is evident that there were some differences in the Ct values shown inB, even though the same number of cells was present in all qPCRs. Thisis however leveled when the values are normalized in respect to theirspecific probes. QPCR was run in duplicates, here showing mean±range ofduplicates.

FIG. 12 : Schematic presentations of the number of specific 1OSDNA-barcode reads mapped to seven different samples as identified bytheir respective sample-ID barcodes (cf. Example 3). A BC samplecontaining a 5% HLA-B0702 CMV pp65 TPR T cell response (correspondingDetection Molecule encoded by barcode 88) were spiked into a HLA-B0702negative BC in fivefold dilutions, creating seven samples with theoreticfrequencies of 5%, 1%, 0.2%, 0.04%, 0.008%, 0.0016% and 0.00032% ofthese T cells. The BC that these cells were spiked into contained ˜0.1%HLA-A1101 EBV-EBNA4 specific T cells (corresponding Detection Moleculeencoded by barcode 4). Samples were stained with the same librarycomprised of 110 different 1OS-labeled Detection Molecules. Each panel,named according to the theoretic frequency of the HLA-B0702 CMVresponse, represents the respective reads mapped to that given sample-IDbarcode. The bars indicate reads mapped to a given 1OS barcode (verticallines) after normalization in respect to the Detection Molecule-inputreads mapped to that same 1OS barcode. Experiments were performed induplicate. Here showing mean.

FIG. 13 : Schematic presentations of the number of specific 2OSDNA-barcode reads mapped to seven different samples as identified bytheir respective sample-ID barcodes (cf. Example 4). A BC samplecontaining a 5% HLA-B0702 CMV pp65 TPR T cell response (correspondingDetection Molecule encoded by barcode A3B18) were spiked into aHLA-B0702 negative BC in fivefold dilutions, creating seven samples withtheoretic frequencies of 5%, 1%, 0.2%, 0.04%, 0.008%, 0.0016% and0.00032% of these T cells. The BC that these cells were spiked intocontained ˜1% HLA-A1101 EBV-EBNA4 specific T cells (correspondingDetection Molecule encoded by barcode A1B4). Samples were stained withthe same library comprised of 110 different 2OS-labeled DetectionMolecules. Each panel, named according to the theoretic frequency of theHLA-B0702 CMV response, represents the respective reads mapped to thatgiven sample-ID barcode. The bars indicate reads mapped to a given 2OSbarcode (vertical lines) after normalization in respect to the DetectionMolecule-input reads mapped to that same 2OS barcode. Experiments wereperformed in duplicate. Here showing mean.

FIG. 14 : Schematic presentations of the number of specific 1OS barcodereads mapped to six different samples as identified by their respectivesample-ID barcodes (cf. Example 5). Six BCs were stained with the samelibrary comprised of 110 different 1OS-labeled Detection Molecules. Eachpanel, named according to the donor BC number, represents the respectivereads mapped to that given sample-ID barcode. The bars indicate readsmapped to a given 1OS barcode (vertical lines) after normalization inrespect to the Detection Molecule-input reads mapped to that same 1OSbarcode.

FIG. 15 : Schematic presentations of the number of specific 2OS barcodereads mapped to six different samples as identified by their respectivesample-ID barcodes (cf. Example 6). Six BCs were stained with the samelibrary comprised of 110 different 2OS-labeled Detection Molecules. Eachpanel, named according to the donor BC number, represents the respectivereads mapped to that given sample-ID barcode. The bars indicate readsmapped to a given 2OS barcode (vertical lines) after normalization inrespect to the Detection Molecule-input reads mapped to that same 2OSbarcode.

FIG. 16 : Schematic presentations of the number of specific 2OS barcodereads mapped to 13 different TIL samples as identified by theirrespective sample-ID barcodes along with the reads mapped to theDetection Molecule-input sample (lower panel) (cf. Example 7). 11 TILsamples were stained with the same library comprised of 175 different2OS-labeled Detection Molecules. Each panel represents the respectivereads mapped to that given sample-ID barcode, which corresponds to TILsfrom resected tumor of a given malignant melanoma patient or to theDetection Molecule-input sample. Panel 1-7 and 9-11 represents the readsobtained from a singular amplification and sequencing of enrichedDetection Molecules, i.e. 2OS barcodes, with individual patient samples.Panel 10 k corresponds to the mean reads obtained from three separateamplification-rounds (with individual sample-IDs) of equivalent to10.000 sorted cells (enriched Detection Molecule) from the samepatient-sample (TIL sample 8). Panel 1K corresponds to the readsobtained from amplification of equivalent to 1000 sorted cells (enrichedDetection Molecule) and panel 100 corresponds to the mean reads obtainedfrom three separate amplification-rounds (with individual sample-IDs) ofequivalent to 100 sorted cells (enriched Detection Molecule) from thesame patient-sample (TIL sample 8). The bars indicate reads mapped to agiven 2OS barcode (vertical lines). The reads were not normalized inrespect to the input sample (here mean of triplicate PCRs with uniquesample-IDs), since only a total of ˜5600 reads were obtained in thesequencing reaction (samples are being resequenced). Even so, theresults are encouraging, with 2OS sequences mapping to most of thebarcode identities present in the Detection Molecule library (input) anda trend showing preferential enrichment of certain, but different, 2OSbarcodes across the patient samples. Furthermore, the replicates ofsample 8, based on different cell numbers, shows comparable results. Thenumber of reads of each barcode is reduced in clonality in respect totheir N6 sequence.

FIG. 17 : Schematic representation of an array imaged with afluorescence microscope. Labeled cells are depicted in two areas of thearray, representing two different targets bound by detection moleculeshaving different binding specificities. The binding molecules havelabeled five cells having one specificity and twelve cells havinganother specificity.

DEFINITIONS AND ABBREVIATIONS

As used everywhere herein, the term “a”, “an” or “the” is meant to beone or more, i. e. at least one.

‘Aff.’ is an abbreviation for affinity.

An “amino acid residue” can be a natural or non-natural amino acidresidue linked peptide bonds or bonds different from peptide bonds. Theamino acid residues can be in D-configuration or L-configuration. Anamino acid residue comprises an amino terminal part (NH₂) and a carboxyterminal part (COOH) separated by a central part comprising a carbonatom, or a chain of carbon atoms, at least one of which comprises atleast one side chain or functional group. NH₂ refers to the amino grouppresent at the amino terminal end of an amino acid or peptide, and COOHrefers to the carboxy group present at the carboxy terminal end of anamino acid or peptide. The generic term amino acid comprises bothnatural and non-natural amino acids. Natural amino acids of standardnomenclature as listed in J. Biol. Chem., 243:3552-59 (1969) and adoptedin 37 C.F.R., section 1.822(b)(2) belong to the group of amino acidslisted in the table herein below. Non-natural amino acids are those notlisted in the Table below. Examples of non-natural amino acids are thoselisted e.g. in 37 C.F.R. section 1.822(b)(4), all of which areincorporated herein by reference. Also, non-natural amino acid residuesinclude, but are not limited to, modified amino acid residues, L-aminoacid residues, and stereoisomers of D-amino acid residues.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

Adjuvant: adjuvants are drugs that have few or no pharmacologicaleffects by themselves, but can increase the efficacy or potency of otherdrugs when given at the same time. In another embodiment, an adjuvant isan agent which, while not having any specific antigenic effect initself, can stimulate the immune system, increasing the response to avaccine.

Agonist: agonist as used herein is a substance that binds to a specificreceptor and triggers a response in the cell. It mimics the action of anendogenous ligand that binds to the same receptor.

Antagonist: antagonist as used herein is a substance that binds to aspecific receptor and blocks the response in the cell. It blocks theaction of an endogenous ligand that binds to the same receptor.

Antibodies: As used herein, the term “antibody” means an isolated orrecombinant binding agent that comprises the necessary variable regionsequences to specifically bind an antigenic epitope. Therefore, anantibody is any form of antibody or fragment thereof that exhibits thedesired biological activity, e.g., binding the specific target antigen.Antibodies can derive from multiple species. For example, antibodiesinclude rodent (such as mouse and rat), rabbit, sheep, camel, and humanantibodies. Antibodies can also include chimeric antibodies, which joinvariable regions from one species to constant regions from anotherspecies. Likewise, antibodies can be humanized, that is constructed byrecombinant DNA technology to produce immunoglobulins which have humanframework regions from one species combined with complementaritydetermining regions (CDR's) from a another species' immunoglobulin. Theantibody can be monoclonal or polyclonal. Antibodies can be divided intoisotypes (IgA, IgG, IgM, IgD, IgE, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2,IgM1, IgM2)

Antibodies: In another embodiment the term “antibody” refers to anintact antibody, or a fragment of an antibody that competes with theintact antibody for antigen binding. In certain embodiments, antibodyfragments are produced by recombinant DNA techniques. In certainembodiments, antibody fragments are produced by enzymatic or chemicalcleavage of intact antibodies. Exemplary antibody fragments include, butare not limited to, Fab, Fab′, F(ab′)2, Fv, and scFv. Exemplary antibodyfragments also include, but are not limited to, domain antibodies,nanobodies, minibodies ((scFv-C.sub.H3).sub.2), maxibodies((scFv-C.sub.H2-C.sub.H3).sub.2), diabodies (noncovalent dimer of scFv).

Antigen presenting cell: An antigen-presenting cell (APC) as used hereinis a cell that displays foreign antigen complexed with MHC on itssurface.

Antigenic peptide: Used interchangeably with binding peptide. Anypeptide molecule that is bound or able to bind into the binding grooveof either MHC class 1 or MHC class 2 molecules.

Antigenic polypeptide: Polypeptide that contains one or more antigenicpeptide sequences.

APC: Antigen presenting cell

Aptamer: the term aptamer as used herein is defined as oligonucleic acidor peptide molecules that bind a specific target molecule. Aptamers areusually created by selecting them from a large random sequence pool, butnatural aptamers also exist. Aptamers can be divided into DNA aptamers,RNA aptamers and peptide aptamers.

Avidin: Avidin as used herein is a glycoprotein found in the egg whiteand tissues of birds, reptiles and amphibians. It contains fouridentical subunits having a combined mass of 67,000-68,000 daltons. Eachsubunit consists of 128 amino acids and binds one molecule of biotin.

Binding molecule (BM): The binding molecule comprised in the detectionmolecule of the present invention is any molecule that can specificallyassociate with, recognize and/or bind to a cell or any other entity,such as another molecule, a surface or a biological cell or other typeof cellular entity (e.g. micelle). Example binding molecules areantibodies, MHC- and MHC-peptide complexes, peptides, small organicmolecules, oligonucleotides, any kind of aptamer, proteins,multicomponent complexes comprising 2, 3, 4, 5, 6, 7, 8, or moresubunits, and supramolecular structures.

Biologically active molecule: A biologically active molecule is amolecule having itself a biological activity/effect or is able to inducea biological activity/effect when administered to a biological system.Biologically active molecules include adjuvants, immune targets (e.g.antigens), enzymes, regulators of receptor activity, receptor ligands,immune potentiators, drugs, toxins, cytotoxic molecules, co-receptors,proteins and peptides in general, sugar moieties, lipid groups, nucleicacids including siRNA, nanoparticles, small molecules.

Bioluminescent: Bioluminescence, as used herein, is the production andemission of light by a living organism as the result of a chemicalreaction during which chemical energy is converted to light energy.

Biotin: Biotin, as used herein, is also known as vitamin H or B₇. Niotinhas the chemical formula C₁₀H₁₆N₂O₃S.

Bispecific antibodies: The term bispecific antibodies as used herein isdefined as antibodies that have binding specificities for at least twodifferent antigens. The antibody can also be trispecific ormultispecific.

Bispecific capture molecule: Molecule that have binding specificitiesfor at least two different antigens. The molecule can also betrispecific or multispecific.

Carrier/linker (Li): Carrier and linker may be used interchangeablyherein, whereby a carrier is one type of a linker according to thepresent invention. A carrier as used herein can be any type of moleculethat is directly or indirectly associated with the binding molecule suchas a MHC peptide complex. In this invention, a carrier will typicallyrefer to a functionalized polymer (e.g. dextran) that is capable ofreacting with a binding molecule such as MHC-peptide complexes, thuscovalently attaching the binding molecule to the carrier, or that iscapable of reacting with scaffold molecules (e.g. streptavidin), thuscovalently attaching streptavidin to the carrier; the streptavidin thenmay bind MHC-peptide complexes. Carrier and scaffold may also be usedinterchangeably herein where scaffold typically refers to smallermolecules of a linker or multimerization domain and carrier typicallyrefers to larger molecule and/or cell like structures.

Cell-detection molecule complex: A complex comprising at least onedetection molecule according to the invention and at least one cell.

Chelating chemical compound: Chelating chemical compound, as usedherein, is the process of reversible binding of a ligand to a metal ion,forming a metal complex.

Chemiluminescent: Chemiluminescence, as used herein, is the emission oflight (luminescence) without emission of heat as the result of achemical reaction.

Chromophore: A chromophore, as used herein, is the part of a visiblycoloured molecule responsible for light absorption over a range ofwavelengths thus giving rise to the colour. By extension the term can beapplied to uv or it absorbing parts of molecules.

Coiled-coil polypeptide: Used interchangeably with coiled-coil peptideand coiled-coil structure. The term coiled-coil polypeptide as usedherein is a structural motif in proteins, in which 2-7 alpha-helices arecoiled together like the strands of a rope

Complement protein: Protein of the complement system.

Counting beads: Beads or particles countable in a flow cytometryexperiment. They can be used as internal control beads enabling absolutecell count in a sample.

Covalent binding: The term covalent binding is used herein to describe aform of chemical bonding that is characterized by the sharing of pairsof electrons between atoms. Attraction-to-repulsion stability that formsbetween atoms when they share electrons is known as covalent bonding.

Crosslinking is the process of chemically joining two or more moleculesby a covalent bond. Crosslinking reagents contain reactive ends tospecific functional groups (primary amines, sulfhydryls, etc.) onproteins or other molecules.

Detection molecule: A molecule comprising a binding molecule (BM), alabel (La) and a linker (Li), wherein ‘a’ is meant to comprise at leastone, or one or more.

Diagnosis: The act or process of identifying or determining the natureand cause of a disease or injury through evaluation

Diabodies: The term “diabodies” refers to small antibody fragments withtwo antigen-binding sites, which fragments comprise a heavy-chainvariable domain (VH) connected to a light-chain variable domain (VL) inthe same polypeptide chain (VH-VL). By using a linker that is too shortto allow pairing between the two domains on the same chain, the domainsare forced to pair with the complementary domains of another chain andcreate two antigen-binding sites.

Dendritic cell: The term dendritic cell as used herein is a type ofimmune cells. Their main function is to process antigen material andpresent it on the surface to other cells of the immune system, thusfunctioning as antigen-presenting cells.

Detection: In this invention detection means any method capable ofmeasuring, or determining the presence of, one molecule alone or boundto another molecule.

Dextran: the term dextran as used herein is a complex, branchedpolysaccharide made of many glucose molecules joined into chains ofvarying lengths. The straight chain consists of α1→6 glycosidic linkagesbetween glucose molecules, while branches begin from α1→3 linkages (andin some cases, α1→2 and α1→4 linkages as well).

Diabodies: The term “diabodies” refers to small antibody fragments withtwo antigen-binding sites, which fragments comprise a heavy-chainvariable domain (VH) connected to a light-chain variable domain (VL) inthe same polypeptide chain (VH-VL). By using a linker that is too shortto allow pairing between the two domains on the same chain, the domainsare forced to pair with the complementary domains of another chain andcreate two antigen-binding sites.

Direct detection of T cells: Direct detection of T cells is used hereininterchangeably with direct detection of TCR and direct detection of Tcell receptor. As used herein direct detection of T cells is detectiondirectly of the binding interaction between a specific T cell receptorand a MHC multimer.

DNA: The term DNA (Deoxyribonucleic acid) duplex as used herein is apolymer of simple units called nucleotides, with a backbone made ofsugars and phosphate atoms joined by ester bonds. Attached to each sugaris one of four types of molecules called bases.

DNA duplex: In living organisms, DNA does not usually exist as a singlemolecule, but instead as a tightly-associated pair of molecules. Thesetwo long strands entwine like vines, in the shape of a double helix.

Electrophilic: electrophile, as used herein, is a reagent attracted toelectrons that participates in a chemical reaction by accepting anelectron pair in order to bond to a nucleophile.

Entity-detection molecule complexes: A complex comprising at least onedetection molecule according to the invention and at least one entity.

Enzyme label: enzyme labelling, as used herein, involves a detectionmethod comprising a reaction catalysed by an enzyme.

Entity: An entity according to the present invention is capable of beingrecognized and/or bound by a detection molecule comprising a bindingmolecule as defined herein, to form a complex comprising the entity andthe detection molecule.

Epitope-focused antibody: Antibodies also include epitope-focusedantibodies, which have at least one minimal essential bindingspecificity determinant from a heavy chain or light chain CDR3 from areference antibody, methods for making such epitope-focused antibodiesare described in U.S. patent application Ser. No. 11/040,159, which isincorporated herein by reference in its entirety.

Flow cytometry: The analysis and/or sorting of single cells using a flowcytometer.

Flow cytometer: Instrument that measures cell size, granularity andflourescence due to bound fluorescent molecules as single cells pass ina stream past photodectors. A flow cytometer carry out the measurementsand/or sorting of individual cells.

Fluorescent: the term fluorescent as used herein is to have the abilityto emit light of a certain wavelength when activated by light of anotherwavelength.

Fluorochromes: Fluorochrome, as used herein, is any fluorescent compoundused as a dye to mark e.g. protein with a fluorescent label.

Fluorophore: A fluorophore, as used herein, is a component of a moleculewhich causes a molecule to be fluorescent.

Folding: In this invention folding means in vitro or in vivo folding ofproteins in a tertiary structure.

Fusion antibody: As used herein, the term “fusion antibody” refers to amolecule in which an antibody is fused to a non-antibody polypeptide atthe N- or C-terminus of the antibody polypeptide.

Glycosylated: Glycosylation, as used herein, is the process or result ofaddition of saccharides to proteins and lipids.

Hapten: A residue on a molecule for which there is a specific moleculethat can bind, e.g. an antibody.

Heteroconjugate antibodies are composed of two covalently joinedantibodies. Such antibodies have, for example, been proposed to targetimmune system cells to unwanted cells.

IgG: IgG as used herein is a monomeric immunoglobulin, built of twoheavy chains and two light chains. Each molecule has two antigen bindingsites.

Isolated antibody: The term “isolated” antibody as used herein is anantibody which has been identified and separated and/or recovered from acomponent of its natural environment.

Immunoconjugates: The invention also pertains to immunoconjugatescomprising an antibody conjugated to a cytotoxic agent such as achemotherapeutic agent, toxin (e.g., an enzymatically active toxin ofbacterial, fungal, plant, or animal origin, or fragments thereof), or aradioactive isotope (i.e., a radioconjugate). Enzymatically activetoxins and fragments thereof that can be used include diphtheria Achain, nonbinding active fragments of diphtheria toxin, exotoxin A chain(from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin Achain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins,Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordicacharantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor,gelonin, mitogellin, restrictocin, phenomycin, enomycin, and thetricothecenes. A variety of radionuclides are available for theproduction of radioconjugated antibodies. Conjugates of the antibody andcytotoxic agent are made using a variety of bifunctionalprotein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives ofimidoesters (such as dimethyl adipimidate HCL), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azidocompounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),diisocyanates (such as tolyene 2,6-diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

Immune monitoring: Immune monitoring of the present invention refers totesting of immune status in the diagnosis and therapy of diseases likebut not limited to cancer, immunoproliferative and immunodeficiencydisorders, autoimmune abnormalities, and infectious disease. It alsorefers to testing of immune status before, during and after vaccinationand transplantation procedures.

Immune monitoring process: a series of one or more immune monitoringanalyses

Indirect detection of T cells: Indirect detection of T cells is usedinterchangeably herein with indirect detection of TCR and indirectdetection of T cell receptor. As used herein indirect detection of Tcells is detection of the binding interaction between a specific T cellreceptor and a MHC multimer by measurement of the effect of the bindinginteraction.

Ionophore: ionophore, as used herein, is a lipid-soluble moleculeusually synthesized by microorganisms capable of transporting ions.

Label (La). The label comprised in the detection molecule of the presentinvention is any molecule, atom or signal the identity of which can befound or determined by any means. The label of a detection moleculespecifies the binding molecule that is linked to the label. Examplelabels are nucleic acid labels including oligonucleotides such as DNA,RNA, PNA, LNA; antibodies; any of the elements such as zinc, iron,magnesium, any of the lanthanides; peptides; proteins; and any type oforganic molecules. The identity of the label can be determined by anyappropriate method for the specific type of label, including but notlimited to mass spectrometry, sequencing (e.g. DNA sequencing, peptidesequencing), gel electrophoresis, gel filtration, and many othermethods.

Labelling: Labelling herein means attachment of a label to a molecule.

Lanthanide: lanthanide, as used herein, series comprises the 15 elementswith atomic numbers 57 through 71, from lanthanum to lutetium.

LDA: limiting dilution assay

A ligand is a molecule capable of binding to and forming a complex witha biomolecule to serve a biological purpose.

Linker (Li). The linker comprised in the detection molecule of thepresent invention is a molecular entity and/or bond that connect thebinding molecule and the label of the detection molecule. The linker canbe of any length, such as length 1-10.000 Å (Ångstrøm), and can have anycomposition, including for example an oligonucleotide, peptide, organicmolecule, and more. A linker according to the present inventioncomprises, or is identical to, a carrier molecule (or carrier), amultimerization domain, a scaffold, a connector and/or a backbone, andmay equally be referred to as such herein. The linker in one embodimentcomprises any linker, carrier or carrier molecule, multimerizationdomain or backbone, to which the binding molecule and the label areattached. The attachment may be direct or indirect, and may comprise acovalent or non-covalent binding.

Liposomes: The term liposome as used herein is defined as a sphericalvesicle with a membrane composed of a phospholipid and cholesterolbilayer. Liposomes, usually but not by definition, contain a core ofaqueous solution; lipid spheres that contain no aqueous material arecalled micelles.

Immunoliposomes: The antibodies disclosed herein can also be formulatedas immunoliposomes. Liposomes comprising the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA 82: 3688 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE).

Immuno-profiling: Immuno profiling as used herein defines the profilingof an individual's antigen-specific T-cell repertoire

MHC: Major histocompatibility complex.

MHC I is used interchangeably herein with MHC class I and denotes themajor histocompatibility complex class I.

MHC II is used interchangeably herein with MHC class II and denotes themajor histocompatibility complex class I.

MHC molecule: a MHC molecule as used everywhere herein is defined as anyMHC class I molecule or MHC class II molecule as defined herein.

A “MHC Class I molecule” as used everywhere herein is usedinterchangeably with MHC I molecule and is defined as a molecule whichcomprises 1-3 subunits, including a MHC I heavy chain, a MHC I heavychain combined with a MHC I beta2microglobulin chain, a MHC I heavychain combined with MHC I beta2microglobulin chain through a flexiblelinker, a MHC I heavy chain combined with an antigenic peptide, a MHC Iheavy chain combined with an antigenic peptide through a linker, a MHC Iheavy chain/MHC I beta2microglobulin dimer combined with an antigenicpeptide, and a MHC I heavy chain/MHC I beta2microglobulin dimer combinedwith an antigenic peptide through a flexible linker to the heavy chainor beta2microglobulin. The MHC I molecule chains can be changed bysubstitution of single or by cohorts of native amino acids, or byinserts, or deletions to enhance or impair the functions attributed tosaid molecule.

MHC Class I like molecules (including non-classical MHC Class Imolecules) include CD1d, HLA E, HLA G, HLA F, HLA H, MICA, MIC B,ULBP-1, ULBP-2, and ULBP-3.

A “MHC Class II molecule” as used everywhere herein is usedinterchangeably with MHC II molecule and is defined as a molecule whichcomprises 2-3 subunits including a MHC II alpha-chain and a MHC IIbeta-chain (i.e. a MHC II alpha/beta-dimer), an MHC II alpha/beta dimerwith an antigenic peptide, and an MHC II alpha/beta dimer combined withan antigenic peptide through a flexible linker to the MHC II alpha orMHC II beta chain, a MHC II alpha/beta dimer combined through aninteraction by affinity tags e.g. jun-fos, a MHC II alpha/beta dimercombined through an interaction by affinity tags e.g. jun-fos andfurther combined with an antigenic peptide through a flexible linker tothe MHC II alpha or MHC II beta chain. The MHC II molecule chains can bechanged by substitution of single or by cohorts of native amino acids,or by inserts, or deletions to enhance or impair the functionsattributed to said molecule. Under circumstances where the MHC IIalpha-chain and MHC II beta-chain have been fused, to form one subunit,the “MHC Class II molecule” can comprise only 1 subunit or 2 subunits ifantigenic peptide is also included.

MHC Class II like molecules (including non-classical MHC Class IImolecules) include HLA DM, HLA DO, I-A beta2, and I-E beta2.

“MHC complexes”, “MHC molecules” and “MHC constructs” are usedinterchangeably herein, and—if not further specified—comprises MHCsloaded with or bound to peptides as well as empty MHCs (not loaded withpeptides).

MHC-peptide complex defines any MHC I and/or MHC II molecule combinedwith antigenic peptide.

A “peptide free MHC Class I molecule” is used interchangeably hereinwith “peptide free MHC I molecule” and as used everywhere herein ismeant to be a MHC Class I molecule as defined above with no peptide.

A “peptide free MHC Class II molecule” is used interchangeably hereinwith “peptide free MHC II molecule” and as used everywhere herein ismeant to be a MHC Class II molecule as defined above with no peptide.

Such peptide free MHC Class I and II molecules are also called “empty”MHC Class I and II molecules.

The MHC molecule may suitably be a vertebrate MHC molecule such as ahuman, a mouse, a rat, a porcine, a bovine or an avian MHC molecule.Such MHC complexes from different species have different names. E.g. inhumans, MHC complexes are denoted HLA. The person skilled in the artwill readily know the name of the MHC complexes from various species.

In general, the term “MHC molecule” is intended to include all alleles.By way of example, in humans e.g. HLA A, HLA B, HLA C, HLA D, HLA E, HLAF, HLA G, HLA H, HLA DR, HLA DQ and HLA DP alleles are of interest shallbe included, and in the mouse system, H-2 alleles are of interest shallbe included. Likewise, in the rat system RT1-alleles, in the porcinesystem SLA-alleles, in the bovine system BoLA, in the avian system e.g.chicken—B alleles, are of interest shall be included.

By the terms “MHC complexes” and “MHC multimers” as used herein aremeant such complexes and multimers thereof, which are capable ofperforming at least one of the functions attributed to said complex ormultimer. The terms include both classical and non-classical MHCcomplexes. The meaning of “classical” and “non-classical” in connectionwith MHC complexes is well known to the person skilled in the art.Non-classical MHC complexes are subgroups of MHC-like complexes. Theterm “MHC complex” includes MHC Class I molecules, MHC Class IImolecules, as well as MHC-like molecules (both Class I and Class II),including the subgroup non-classical MHC Class I and Class II molecules.

MHC multimer: The terms MHC multimer, MHC-multimer, MHCmer and MHC'merherein are used interchangeably, to denote a complex comprising morethan one MHC-complex and/or MHC-peptide complex, held together bycovalent or non-covalent bonds. In one embodiment a MHC multimercomprises 2 copies (dimer), 3 copies (trimer), 4 copies (tetramer), 5copies, (pentamer), 6 copies (hexamer), 7 copies (heptamer), 8 copies(octamer), 9 copies (nonamer), 10 copies (decamer), 11 copies, 12copies, 13 copies, 14 copies, 15 copies, 16 copies, 17 copies, 18copies, 19 copies or 20 copies,

Monoclonal antibodies: Monoclonal antibodies, as used herein, areantibodies that are identical because they were produced by one type ofimmune cell and are all clones of a single parent cell.

Monovalent antibodies: The antibodies in the present invention can bemonovalent antibodies. Methods for preparing monovalent antibodies arewell known in the art. For example, one method involves recombinantexpression of immunoglobulin light chain and modified heavy chain. Theheavy chain is truncated generally at any point in the Fc region so asto prevent heavy chain crosslinking. Alternatively, the relevantcysteine residues are substituted with another amino acid residue or aredeleted so as to prevent crosslinking. In vitro methods are alsosuitable for preparing monovalent antibodies. Digestion of antibodies toproduce fragments thereof, particularly, Fab fragments, can beaccomplished using routine techniques known in the art.

Multimerization domain: A multimerization domain is a type of linkeraccording to the present invention. It may also be used as a synonym tolinker. It defines a molecule, a complex of molecules, or a solidsupport, to which one or more binding molecules, such as MHC orMHC-peptide complexes, can be attached. A multimerization domain consistof one or more carriers and/or one or more scaffolds and may alsocontain one or more scaffold molecules (or connectors) connectingcarrier to scaffold, carrier to carrier, scaffold to scaffold. Themultimerization domain may also contain one or more scaffold moleculesthat can be used for attachment of binding molecules and/or othermolecules to the multimerization domain. Multimerization domains thuscomprises IgG, streptavidin, streptactin, micelles, cells, polymers,beads and other types of solid support, and small organic moleculescarrying reactive groups or carrying chemical motifs that can bindbinding molecules such as MHC complexes and other molecules.

Nanobodies: Nanobodies as used herein is a type of antibodies derivedfrom camels, and are much smaller than traditional antibodies.

Neutralizing antibodies: neutralizing antibodies as used herein is anantibody which, on mixture with the homologous infectious agent, reducesthe infectious titer.

NMR: NMR (Nuclear magnetic resonance), as used herein, is a physicalphenomenon based upon the quantum mechanical magnetic properties of anatom's nucleus. NMR refers to a family of scientific methods thatexploit nuclear magnetic resonance to study molecules.

Non-covalent: The term non-covalent bond as used herein is a type ofchemical bond that does not involve the sharing of pairs of electrons,but rather involves more dispersed variations of electromagneticinteractions.

Nucleic acid duplex: A nucleic acid is a complex, high-molecular-weightbiochemical macromolecule composed of nucleotide chains that conveygenetic information. The most common nucleic acids are deoxyribonucleicacid (DNA) and ribonucleic acid (RNA).

Nucleophilic: a nucleophile, as used herein, is a reagent that forms achemical bond to its reaction partner (the electrophile) by donatingboth bonding electrons.

“One or more” as used everywhere herein is intended to include one and aplurality i.e. more than one.

Pegylated: pegylated, as used herein, is conjugation of Polyethyleneglycol (PEG) to proteins.

Peptide or protein: Any molecule composed of at least two amino acids.Peptide normally refers to smaller molecules of up to around 30 aminoacids and protein to larger molecules containing more amino acids.

Phosphorylated; phosphorylated, as used herein, is the addition of aphosphate (PO₄) group to a protein molecule or a small molecule.

PNA: PNA (Peptide nucleic acid) as used herein is a chemical similar toDNA or RNA. DNA and RNA have a deoxyribose and ribose sugar backbone,respectively, whereas PNA's backbone is composed of repeatingN-(2-aminoethyl)-glycine units linked by peptide bonds. The variouspurine and pyrimidine bases are linked to the backbone by methylenecarbonyl bonds. PNAs are depicted like peptides, with the N-terminus atthe first (left) position and the C-terminus at the right.

“A plurality” as used everywhere herein should be interpreted as two ormore.

This applies i.a. to the MHC complex and the binding entity. When aplurality of MHC complexes is attached to the multimerization domain,such as a scaffold or a carrier molecule, the number of MHC complexesneed only be limited by the capacity of the multimerization domain.

Polyclonal antibodies: a polyclonal antibody as used herein is anantibody that is derived from different B-cell lines. They are a mixtureof immunoglobulin molecules secreted against a specific antigen, eachrecognising a different epitope.

Polymer: the term polymer as used herein is defined as a compoundcomposed of repeating structural units, or monomers, connected bycovalent chemical bonds.

Polypeptide: Peptides are the family of short molecules formed from thelinking, in a defined order, of various α-amino acids. The link betweenone amino acid residue and the next is an amide bond and is sometimesreferred to as a peptide bond. Longer peptides are referred to asproteins or polypeptide.

Polysaccharide: The term polysaccharide as used herein is defined aspolymers made up of many monosaccharides joined together by glycosidiclinkages.

Radicals: radicals, as used herein, are atomic or molecular species withunpaired electrons on an otherwise open shell configuration. Theseunpaired electrons are usually highly reactive, so radicals are likelyto take part in chemical reactions.

Radioactivity: Radioactive decay is the process in which an unstableatomic nucleus loses energy by emitting radiation in the form ofparticles or electromagnetic waves. RNA: RNA (Ribonucleic acid) as usedherein is a nucleic acid polymer consisting of nucleotide monomers thatplays several important roles in the processes that translate geneticinformation from deoxyribonucleic acid (DNA) into protein products.

RNA: RNA (Ribonucleic acid) as used herein is a nucleic acid polymerconsisting of nucleotide monomers that plays several important roles inthe processes that translate genetic information from deoxyribonucleicacid (DNA) into protein products

Scaffold: A scaffold is typically an organic molecule carrying reactivegroups, capable of reacting with reactive groups on a MHC- orMHC-peptide complex. Particularly small organic molecules of cyclicstructure (e.g. functionalized cycloalkanes or functionalized aromaticring structures) are termed scaffolds. Scaffold and carrier are usedinterchangeably herein where scaffold typically refers to smallermolecules of a multimerization domain and carrier typically refers tolarger molecule and/or cell like structures. Both scaffold, carrier andmultimerization domain are types of linkers according to the presentinvention.

Sequencing. In the present aspect it is understood that sequencingencompasses all types of sequencing of a given nucleic acid sequence,including also e.g. deep-sequencing or next-generation sequencing, inwhich amplified barcode sequences of a nucleic acid label (the PCRproduct) is sequenced a large number of repetitive time (number of totalreads, e.g. 100.000s of reads). The number of reads for the individualbarcode sequence will relate to their quantitative presence in theamplification product, which again represents their quantitativepresence before amplification, since all DNA-barcodes have similaramplification properties. Thus, the number of reads for a specificbarcode sequence compared to the total number of reads will correlate tothe presence of antigen responsive cells in the test-sample.

Staining: In this invention staining means specific or unspecificlabelling of cells by binding labeled molecules to defined proteins orother structures on the surface of cells or inside cells. The cells areeither in suspension or part of a tissue. The labeled molecules can beMHC multimers, antibodies or similar molecules capable of bindingspecific structures on the surface of cells.

Streptavidin: Streptavidin as used herein is a tetrameric proteinpurified from the bacterium Streptomyces avidinii. Streptavidin iswidely use in molecular biology through its extraordinarily strongaffinity for biotin.

Sugar: Sugars as used herein include monosaccharides, disaccharides,trisaccharides and the oligosaccharides—comprising 1, 2, 3, and 4 ormore monosaccharide units respectively.

TCR: T-Cell Receptor

Therapy: Treatment of Illness or Disability

Vaccine: A vaccine is an antigenic preparation used to establishimmunity to a disease or illness and thereby protect or cure the bodyfrom a specific disease or illness. Vaccines are either prophylactic andprevent disease or therapeutic and treat disease. Vaccines may containmore than one type of antigen and is then called a combined vaccine.

Vaccination: The introduction of vaccine into the body of human oranimals for the purpose of inducing immunity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a detection molecule comprising a bindingmolecule (BM), a linker (Li) and a label (La). Said detection moleculecan be used in various methods, such as detection methods as outlinedherein.

The binding molecules of the invention are capable of specificallyassociating with, such as recognizing and/or binding to, a target entityor cell.

The present invention provides a means for detecting detection moleculescapable of binding to—and/or bound to—individual cells or to a subset ofcells. Alternatively, the present invention provides a means fordetecting the presence and/or abundance of cells capable of binding todetection molecules.

The present invention is in one embodiment used for epitope discovery,analytical studies, diagnosis, and therapy, and may be used in vivo(e.g. in animals) or in vitro (e.g. in any kind of cell samples such asblood, synovial fluid or bone marrow).

The present invention is in one embodiment used to characterize cellsfor their ability to bind certain detection molecules. The presentinvention is in one embodiment used to detect the presence of certaincell types, identified by their detection molecule-binding pattern.

In another embodiment, the technology is used for T-cell epitopemapping, immune-recognition discovery, or measuring immune reactivityafter vaccination or immune-related therapies.

Composition

It is an aspect of the present invention to provide a detection moleculecomprising

-   -   a. a binding molecule (BM),    -   b. a linker (Li), and    -   c. a label (La).

The components of the detection molecule namely binding molecule, linkerand label are defined herein elsewhere. It is understood that ‘a’ in thepresent context include at least one, or one or more, of each of saidcomponents. A binding molecule can thus be a monomeric or a multimericbinding molecule.

The present invention thus provides a detection molecule comprising atleast one binding molecule (BM), at least one linker (Li) and at leastone label (La).

It is understood that each component of the detection molecule, namelythe binding molecule, the linker and the label, individually can beselected from any of the binding molecules, linkers and labels disclosedherein throughout. Thus, any combination of binding molecules, linkersand labels are encompassed within the present disclosure and invention.

It is also an aspect of the present invention to provide anentity-detection molecule complex, wherein said complex comprises

-   -   a. at least one detection molecule comprising a binding molecule        (BM), a linker (Li) and a label (La), and    -   b. at least one entity.

It is also an aspect of the present invention to provide acell-detection molecule complex, wherein said complex comprises

-   -   c. at least one detection molecule comprising a binding molecule        (BM), a linker (Li) and a label (La), and    -   d. at least one cell.

In one embodiment the cell-detection molecule complexes comprises acell, such as an immune cell, associated with or bound to a detectionmolecule having a binding molecule specific for the immune cell.

In one embodiment one or more components of the detection molecule; thatis one or more of the binding molecule, the linker and the label areeach as described in WO 2002/072631, WO 2009/106073 or WO 2009/003492,which are hereby incorporated by reference in their entirety.

In a particular embodiment the detection molecule has one or morecharacteristics as the detection molecules described in, and/or isprepared as described in, WO 2009/003492; European Journal ofImmunology, vol. 31, pp 32-38, January 2001 (Hansen et al); or Journalof Immunological Methods, vol. 241, issues 1-2, 31 Jul. 2000 (Le Doussalet al).

In an aspect of the present invention the label will serve as a specificlabel for identifying a given binding molecule.

In an aspect of the present invention the label, such as a nucleic acidlabel, will serve as a specific label for a given binding molecule, suchas a MHC molecule or peptide-MHC molecule that is multimerized to form aMHC multimer.

In one embodiment the present invention provides a detection moleculecomprising

-   -   a. a monomeric or a multimeric major histocompatibility complex        (MHC) molecule, such as a monomeric or multimeric peptide MHC        complex,    -   b. a linker comprising a multimerization domain and optionally        one or more connectors, and    -   c. a nucleic acid label.

In one embodiment the present invention provides a detection moleculecomprising

-   -   a. a monomeric or a multimeric major histocompatibility complex        (MHC) molecule, such as a monomeric or multimeric peptide MHC        complex,    -   b. a linker comprising a multimerization domain and optionally        one or more connectors, and    -   c. a peptide label.

In one embodiment the present invention provides a detection moleculecomprising

-   -   a. an anti-target molecule capable of associating with,        recognizing and/or binding to a predetermined marker molecule        (or target) on a cell type, wherein said marker molecule is        specific for a certain cell type    -   b. a linker (Li) comprising a multimerization domain and        optionally one or more connectors, and    -   c. a nucleic acid label.

Anti-target molecules are defined in more detail herein below.

In one embodiment the present invention provides a detection moleculecomprising

-   -   a. CD1, wherein said CD1 is selected from the group consisting        of CD1 CD1a, CD1b, CD1c, CD1d and CD1e,    -   b. a linker comprising a multimerization domain and optionally        one or more connectors, and    -   c. a nucleic acid label.

In one the linker of the detection molecule comprises a multimerizationdomain, such as a polysaccharide such as dextran, and optionally furthercomprises streptavidin or avidin whereby the binding molecule and/or thelabel comprises biotin.

In one embodiment the invention relates to a multimeric majorhistocompatibility complex (MHC) comprising

-   -   a. two or more MHC molecules linked by a backbone molecule        (linker); and    -   b. at least one nucleic acid molecule (label) linked to said        backbone, wherein said nucleic acid molecule comprises a central        stretch of nucleic acids (barcode region) designed to be        amplified by e.g. PCR.

In one embodiment the invention relates to a major histocompatibilitycomplex (MHC) comprising

-   -   a. one MHC molecule    -   b. a backbone molecule (linker); and    -   c. at least one nucleic acid molecule (label) linked to said        backbone, wherein said nucleic acid molecule comprises a central        stretch of nucleic acids (barcode region) designed to be        amplified by e.g. PCR.

The invention provides a composition comprising one detection molecule,or one set of identical detection molecules.

Also provided is a composition comprising two or more differentdetection molecules, or two or more sets of different detectionmolecules, each detection molecule comprising at least one bindingmolecule (BM), at least one linker (Li) and at least one label (La) asdefined herein,

wherein each of the two or more detection molecules, or two or more setsof detection molecules, comprises a label which is unique to andspecific for the binding molecule of each of said two or more differentdetection molecules.

In one embodiment said composition comprising two or more differentdetection molecules, or two or more sets of different detectionmolecules, comprises 2 to 1,000,000 different detection molecules, suchas 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40 different detection molecules; for example 1-3, 3-5, 5-10, 10-15,15-20, 20-25, 25-30, 30-35, 35-40, 40-50, 50-60, 60-70, 70-80, 80-90,90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-175, 175-200,200-250, 250-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900,900-1000, 1000-1500, 1500-2000, 2000-3000, 3000-4000, 4000-5000,5000-7500, 7500-10,000, 10,000-20,000, 20,000-50,000, 50,000-100,000,100,000-200,000, 200,000-500,000, 500,000-1,000,000 different detectionmolecules, or sets of different detection molecules.

The term ‘different detection molecules’ as used herein means that oneor more of the binding molecule and/or label of each of the two or moredetection molecules, or two or more sets of detection molecules, aredifferent. Thus, the different detection molecules have differentbinding specificities (targets) and/or have different labels.

Another embodiment of the present invention relates to a compositioncomprising a subset of multimeric major histocompatibility complexes(MHC's) according to the invention, wherein each set of MHC's has adifferent peptide decisive for T cell recognition and a unique “barcode”region in the nucleic acid label (such as a DNA molecule).

Ratio of Binding Molecule/Label

In one embodiment there is a one-to-one relation between the label andthe binding molecule, i.e. only one specific label (e.g. a specificoligonucleotide sequence) is attached to a specific binding molecule(e.g. a specific antibody). In other words, identifying the label (e.g.by sequencing an oligonucleotide label) will unambiguously identify thebinding molecule attached to the label.

In another embodiment there is a one-to-one relation between the labeland a group of binding molecules, i.e. only one specific label (e.g. aspecific oligonucleotide sequence) is attached to a specific set ofbinding molecules (e.g. twenty different antibodies). In other words,identifying the label (e.g. by sequencing an oligonucleotide label) willunambiguously identify the group of binding molecule that may beattached to the label; identifying the label, however, will not identifythe specific binding molecule that is attached to this particular copyof the label.

In yet another embodiment there is a one-to-one relation between a groupof labels and a group of binding molecules, i.e. the label chosen for aspecific detection molecule is chosen from a set of labels (e.g. twentydifferent oligonucleotide sequences) and the binding molecule chosen fora specific detection molecule is chosen from a set of binding molecules(e.g. twenty different antibodies). In other words, identifying thelabel of a detection molecule (e.g. by sequencing an oligonucleotidelabel) will unambiguously identify the group of binding molecule thatmay be attached to the label; identifying the label, however, will notidentify the specific binding molecule that is attached to thisparticular copy of the label.

In another embodiment there is a one-to-one relation between a group oflabels and a binding molecule, i.e. the label chosen for a specificdetection molecule is chosen from a set of labels (e.g. twenty differentoligonucleotide sequences). In other words, identifying the label of adetection molecule (e.g. by sequencing an oligonucleotide label) willunambiguously identify the binding molecule attached to the label;identifying the binding molecule, however, will not identify thespecific label that is attached to this particular copy of the label. Incases where a certain label itself binds to the cells (and thus mightlead to the wrong conclusion that the attached binding molecule binds tothe cell), using several labels to encode the same binding molecule willallow the operator to distinguish binding of the binding molecule frombinding of the label to the cell. Using several labels for the samebinding molecule, or using several binding molecules for the same labelcan thus be used as internal controls.

Label

The label comprised in the detection molecule of the present inventionis any molecule, atom or signal the identity of which can be found ordetermined by any means. The label of a detection molecule specifies theidentity of the binding molecule that is linked to the label. Theidentity of the label can be determined by any appropriate method forthe specific type of label.

In one embodiment there is provided a detection molecule comprising oneor more binding molecules, one or more linkers, and one or more labels.The one or more labels may be connected with the other components of thedetection molecule in any manner. In one embodiment, one or more labelsare connected to the linker (or one of the linkers, or both or all ofthe multiple linkers) of the detection molecule. In one embodiment oneor more labels are connected to the binding molecule (or one of thebinding molecules, or both or all of the multiple binding molecules) ofthe detection molecule.

In one embodiment each detection molecule comprises one label. Inanother embodiment the detection molecule comprises two or more labels,such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20labels. In one embodiment the detection molecule comprises 1-2, 2-3,3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-15, 15-20, 20-25, 25-30, 30-40,40-50, 50-75, 75-100, 100-150, 150-200, 200-250, 250-500, 500-750,750-1000 labels.

In one embodiment each of said labels are identical to each other. Inanother embodiment each of said labels are different from each other. Inyet another embodiment two or more of said labels are different withrespect to each other.

In one embodiment the label linked to a given detection molecule isdifferent from the label linked to other detection molecules used in thesame assay.

In another embodiment the detection molecule comprises two differenttypes of labels, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20 different types of labels.

Labeling Molecules

Labelling molecules are molecules that can be detected in a certainanalysis, i.e. the labelling molecules provide a signal detectable bythe used method. Label and labelling molecules are used interchangeablyherein.

The labelling molecule may be any labelling molecule suitable for director indirect detection. By the term “direct” is meant that the labellingmolecule can be detected per se without the need for a secondarymolecule, i.e. is a “primary” labelling molecule. By the term “indirect”is meant that the labelling molecule can be detected by using one ormore “secondary” molecules, i.e. the detection is performed by thedetection of the binding of the secondary molecule(s) to the primarymolecule.

In one embodiment the labelling molecule is attached to the linker

In one embodiment the labelling molecule is attached to the bindingmolecule.

The labelling molecule in one embodiment comprises a suitable linker or‘connector molecule’ for attachment to the linker. Linkers suitable forattachment to labelling molecules would be readily known by the personskilled in the art and as described elsewhere herein.

A label of the present invention is in one embodiment selected from thegroup consisting of polymers, nucleic acids, oligonucleotides, peptides,fluorescent labels, phosphorescent labels, enzyme labels,chemiluminescent labels, bioluminescent labels, haptens, antibodies,dyes, nanoparticle labels, elements, metal particles, heavy metallabels, isotope labels, radioisotopes, stable isotopes, chains ofisotopes and single atoms.

Labels may be organic or inorganic molecules or particles.

Organic molecules labels include ribonucleic acids (e.g. RNA, DNA orunnatural DNA, RNA, and XNA (e.g. PNA, LNA, GNA, TNA) andmononucleotides, peptides and other polyamides (e.g. peptides comprisingβ-amino acid residues), lipids, carbohydrates, amino acids, and manyother molecules.

Inorganic molecule labels include the elements (e.g. Lanthanum, Cerium,Praseodymium, Neodymium, Promethium, Samarium, Europium, Gadolinium,Terbium, Dysprosium, and the rest of the elements known). The elementsmay coupled to the linker by way of chelates that coordinate the ions(interact non-covalently with the ions), where the chelates are thenlinked to the linker (in cases such as Gadolinium where the element canexist on ionic form), or the element may be contained in micelles. Forsome applications, rare elements are particularly favorable. For otherapplications heavy metals are particularly favorable.

Isotopes may also be used as labels (e.g. Carbon isotopes ¹²C, ¹³C and¹⁴C). Furthermore, label molecules comprising combinations of differentisotopes and elements may also be used.

Particle labels include quantum dots, nanoparticles, micelles (e.g.comprising different fluorophores internally or in its membrane) andother particulate structures.

A molecule label may have a molecular weight of between 1 Da and severalmillion Da. In some instances a very low molecular weight is preferred,such as a molecular weight of 1-10 Da, 11-50 Da, 50-250 Da, or 251-500Da. This may for example be the case when mass spectrometry is used todetect the identity of element labels (e.g. Gadolinium, Gd). In othercases a low molecular weight, e.g. 501-2000 Da, 2001-5000 Da, or5001-10000 Da may be preferred. This may be the case when e.g. peptidelabels are used, where the peptide label comprises around 10-40 aminoacid residues. In yet other cases, a high molecular weight of themolecule label is practical, and the molecular weight of the moleculelabel may be 10001-50000 Da, 50001-200000 Da, or 200000-1000000 Da. Thismay be the case e.g. in cases where a ribonucleic acid label is used,where the coding region (also called the barcode region or barcodesequence) is of significant length (e.g. 10-20 nucleotides) and where itis practical to have flanking primer binding regions of each 10-20nucleotides, plus other sequences of different practical use. Theresulting oligonucleotide label may in these cases be 30-1000 nt long,corresponding to molecular weights of about 10000-600000 Da. Finally,multi-molecule structures, such as in cases where a number of differentfluorescent proteins are ordered in an array by binding to specificregions in a template DNA, where the total label thus comprises a longoligonucleotide to which is bound a number of proteins, and the totalmolecular weight of the label may thus be 50000-200000 Da,200001-100000, or 1000001-10000000 Da.

In one embodiment the labels are fluorophores and other molecules thatemit or absorb radiation. The fluorophores and other molecules emittingor absorbing radiation may be of organic or inorganic nature, and can bee.g. small molecules as well as large proteins. In one embodiment, it isparticularly favorable if all the fluorophores and other molecules thatemit or absorb radiation are within the same narrow range of emissionwavelength optimum, such as having wavelength optima in the range 1-10nm, 11-30 nm, 31-100 nm, 101-200 nm, 201-300 nm, 301-400 nm, 401-500 nm,501-600 nm, 601-700 nm, 701-800 nm, 800-900 nm, 901-1200 nm, 1201-1500nm, or larger than 1500 nm. As an example, if the instrument has anarrow range of wavelengths that can be detected, it is advantageousthat all labels fall within this range of detection. On the other hand,if the instrument used to detect the radiation emitted by the labels hasa wide span of detectable wavelengths, it is desirable that thedifferent labels used in an experiment fall in several of theabove-mentioned ranges, as this will result in little overlap betweenemission of different labels, and therefore more accurate detection ofrelative abundance of the different labels of an experiment. Emittedradiation may be phosphorescence, luminescence, fluorescence and more.

The labelling compound may suitably be selected:

from fluorescent labels such as 5-(and 6)-carboxyfluorescein, 5- or6-carboxy-fluorescein, 6-(fluorescein)-5-(and 6)-carboxamido hexanoicacid, fluorescein isothio-cyanate (FITC), rhodamine,tetramethylrhodamine, and dyes such as Cy2, Cy3, and Cy5, optionallysubstituted coumarin including AMCA, PerCP, phycobiliproteins includingR-phycoerythrin (RPE) and allophycoerythrin (APC), Texas Red, PrincestonRed, Green fluorescent protein (GFP) and analogues thereof, andconjugates of R-phycoerythrin or allophycoerythrin and e.g. Cy5 or TexasRed, and inorganic fluorescent labels based on semiconductornanocrystals (like quantum dot and Qdot™ nanocrystals), andtime-resolved fluorescent labels based on lanthanides like Eu3+ andSm3+,

from haptens such as DNP, biotin, and digoxiginin,

from enzymatic labels such as horse radish peroxidase (HRP), alkalinephosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphatedehydrogenase, beta-N-acetyl-glucosaminidase, β-glucuronidase,invertase, Xanthine Oxidase, firefly luciferase and glucose oxidase(GO),

from luminiscence labels such as luminol, isoluminol, acridinium esters,1,2-dioxetanes and pyridopyridazines,

from radioactivity labels such as incorporated isotopes of iodide,cobalt, selenium, tritium, and phosphor, and

from single atoms such as zinc (Zn), iron (Fe), magnesium (Mg), any ofthe lanthanides (Ln) including La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy,Ho, Er, Tm, Yb and Lu; scandium (Sc) and yttrium (Y).

Different principles of labelling and detection exist, based on thespecific property of the labelling molecule. Examples of different typesof labelling are emission of radioactive radiation (radionuclide,isotopes), absorption of light (e.g. dyes, chromophores), emission oflight after excitation (fluorescence from fluorochromes), NMR (nuclearmagnetic resonance form paramagnetic molecules) and reflection of light(scatter from e.g. such as gold-, plastic- or glass-beads/particles ofvarious sizes and shapes).

Alternatively, the labelling molecules can have an enzymatic activity,by which they catalyze a reaction between chemicals in the nearenvironment of the labelling molecules, producing a signal, whichinclude production of light (chemi-luminescence), precipitation ofchromophor dyes, or precipitates that can be detected by an additionallayer of detection molecules. The enzymatic product can deposit at thelocation of the enzyme or, in a cell based analysis system, react withthe membrane of the cell or diffuse into the cell to which it isattached. Examples of labelling molecules and associated detectionprinciples are shown in the table below.

Labelling substance Effect Assay-principle Fluorochromes emission oflight having a ^(¤)Photometry, Microscopy, specific spectra spectroscopyPMT, photographic film, CCD's (Color-Capture Device or Charge-coupleddevice). Radionuclide irradiation, α, 

 or gamma Scintillation counting, GM-

 rays tube, photographic film, excitation of phosphor-imager screenEnzyme; catalysis of H₂O₂ reduction ^(¤)Photometry, Microscopy, HRP,(horse reddish using luminol as Oxygen spectroscopy peroxidase),acceptor, resulting in oxidized PMT, photographic film, peroxidases ingeneral luminal + light CCD's (Colour-Capture catalysis of H₂O₂reduction Device or Charge-coupled using a soluble dye, or device),molecule containing a hapten, Secondary label linked such as a biotinresidue as antibody Oxygen acceptor, resulting in precipitation. Thehabten can be recognized by a detection molecule. Particles; gold,polystyrene Change of scatter, reflection Microscopy, cytometry, beads,pollen and other and transparency of the electron microscopy particlesassociated entity PMT's, light detecting devices, flowcytometry scatterAP (Alkaline Phosphatase) Catalyze a chemical ^(¤)Photometry,Microscopy, conversion of a non- spectroscopy detectable to aprecipitated Secondary label linked detectable molecule, such asantibody a dye or a hapten Ionophores or chelating Change in absorptionand ^(¤)Photometry, Cytometry, chemical compounds binding emissionspectrums when spectroscopy to specific ions, e.g. Ca²⁺ binding. Changein intensity Lanthanides Fluorescence ^(¤)photometry, cytometry,Phosphorescence spectroscopy Paramagnetic NMR (Nuclear magneticresonance) DNA fluorescing stains Propidium iodide ^(¤)Photometry,cytometry, Hoechst stain spectroscopy DAPI AMC DraQ5 ™ Acridine orange7-AAD Nucleic acid label Sequence, mass PCR amplification, sequeningMass spec Gel electrophoresis, PAGE QPCR Peptide label Sequence, massSequencing Labelling substance Effect Assay-principle Mass spec Gelelectrophoresis, PAGE

Labelling molecules can be attached to a given binding molecule bycovalent linkage as described for attachment of binding molecules tomultimerization domains elsewhere herein. The attachment can be directlybetween reactive groups in the labelling molecule and reactive groups inthe binding molecule or the attachment can be through a linkercovalently attached to labelling molecule and binding molecule. Whenlabelling MHC multimers the label can be attached either to the MHCcomplex (heavy chain, β2m or peptide) or to the multimerization domain.

In particular,

one or more labelling molecules may be attached to the carrier molecule,or one or more labelling molecules may be attached to one or more of thescaffolds, or one or more labelling compounds may be attached to one ormore of the binding molecules, or one or more labelling compounds may beattached to the carrier molecule and/or one or more of the scaffoldsand/or one or more of the binding molecules.

A single labelling molecule does not always generate sufficient signalintensity. The signal intensity can be improved by assembling singlelabel molecules into large multi-labelling compounds, containing two ormore label residues. Generation of multi-label compounds can be achievedby covalent or non-covalent, association of labelling molecules with amajor structural molecule. Examples of such structures are synthetic ornatural polymers (e.g. dextramers), proteins (e.g. streptavidin), orpolymers. The labelling molecules in a multi-labelling compound can allbe of the same type or can be a mixture of different labellingmolecules.

Detection principles can be applied to flow cytometry, stationarycytometry, and batch-based analysis. Most batch-based approaches can useany of the labelling substances depending on the purpose of the assay.Flow cytometry primarily employs fluorescence, whereas stationarycytometry primarily employs light absorption, e.g. dyes or chromophoredeposit from enzymatic activity.

In flow cytometry the typical label is detected by its fluorescence.Most often a positive detection is based on the presence of light from asingle fluorochrome, but in other techniques the signal is detected by ashift in wavelength of emitted light; as in FRET based techniques, wherethe exited fluorochrome transfer its energy to an adjacent boundfluorochrome that emits light, or when using Ca²⁺ chelating fluorescentprops, which change the emission (and absorption) spectra upon bindingto calcium. Preferable labelling molecules employed in flow cytometryare illustrated in the tables and described in the following.

Simple fluorescent labels:

-   -   Fluor dyes, Pacific Blue™, Pacific Orange™, Cascade Yellow™,    -   AlexaFluor® (AF);        -   AF405, AF488,AF500, AF514, AF532, AF546, AF555, AF568,            AF594, AF610, AF633, AF635, AF647, AF680, AF700, AF710,            AF750, AF800    -   Quantum Dot based dyes, QDot® Nanocrystals (Invitrogen,        MolecularProbs)        -   Qdot®525, Qdot®565, Qdot®585, Qdot®605, Qdot®655, Qdot®705,            Qdot®800    -   DyLight™ Dyes (Pierce) (DL);        -   DL549, DL649, DL680, DL800    -   Fluorescein (Flu) or any derivate of that, ex. FITC    -   Cy-Dyes        -   Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7    -   Fluorescent Proteins;        -   RPE, PerCp, APC        -   Green fluorescent proteins;            -   GFP and GFP-derived mutant proteins; BFP, CFP, YFP,                DsRed, T1, Dimer2, mRFP1, MBanana, mOrange, dTomato,                tdTomato, mTangerine, mStrawberry, mCherry    -   Tandem dyes:        -   RPE-Cy5, RPE-Cy5.5, RPE-Cy7, RPE-AlexaFluor® tandem            conjugates; RPE-Alexa610, RPE-TxRed        -   APC-Aleca600, APC-Alexa610, APC-Alexa750, APC-Cy5, APC-Cy5.5    -   Ionophors; ion chelating fluorescent props        -   Props that change wavelength when binding a specific ion,            such as Calcium Props that change intensity when binding to            a specific ion, such as Calcium    -   Combinations of fluorochromes on the same marker. Thus, the        marker is not identified by a single fluorochrome but by a code        of identification being a specific combination of fluorochromes,        as well as inter related ratio of intensities.    -   Example: Antibody Ab1 and Ab2, are conjugated to both. FITC and        BP but Ab1 have 1 FITC to 1 BP whereas Ab2 have 2 FITC to 1 BP.        Each antibody may then be identified individually by the        relative intensity of each fluorochrome. Any such combinations        of n fluorochromes with m different ratios can be generated.

Examples of preferable fluorochromes:

Excitation Emission Fluorofor/Fluorochrome nm nm2-(4′-maleimidylanilino)naphthalene-6- 322 417 sulfonic acid, sodiumsalt 5-((((2-iodoacetyl)amino)ethyl)amino) 336 490naphthalene-1-sulfonic acid Pyrene-1-butanoic acid 340 376 AlexaFluor350 (7-amino-6-sulfonic acid-4- 346 442 methyl coumarin-3-acetic acid)AMCA (7-amino-4-methyl coumarin-3- 353 442 acetic acid)7-hydroxy-4-methyl coumarin-3-acetic acid 360 455 Marina Blue(6,8-difluoro-7-hydroxy-4- 362 459 methyl coumarin-3-acetic acid)7-dimethylamino-coumarin-4-acetic acid 370 459 Fluorescamin-N-butylamine adduct 380 464 7-hydroxy-coumarine-3-carboxylic acid 386 448CascadeBlue (pyrene-trisulphonic acid 396 410 acetyl azide) CascadeYellow 409 558 Pacific Blue (6,8 difluoro-7-hydroxy 416 451coumarin-3-carboxylic acid) 7-diethylamino-coumarin-3-carboxylic acid420 468 N-(((4-azidobenzoyl)amino)ethyl)-4- 426 534amino-3,6-disulfo-1,8-naphthalimide, dipotassium salt Alexa Fluor 430434 539 3-perylenedodecanoic acid 440 4488-hydroxypyrene-1,3,6-trisulfonic acid, 454 511 trisodium salt12-(N-(7-nitrobenz-2-oxa-1,3-diazol-4- 467 536 yl)amino)dodecanoic acidN,N′-dimethyl-N-(iodoacetyl)-N′-(7- 478 541nitrobenz-2-oxa-1,3-diazol-4- yl)ethylenediamine Oregon Green 488(difluoro carboxy 488 518 fluorescein) 5-iodoacetamidofluorescein 492515 Propidium iodide-DNA adduct 493 636 Carboxy fluorescein 495 519

Examples of preferable fluorochrome families:

Fluorochrome family Example fluorochrome AlexaFluor ®(AF) AF ®350,AF405, AF430, AF488, AF500, AF514, AF532, AF546, AF555, AF568, AF594,AF610, AF633, AF635, AF647, AF680, AF700, AF710, AF750, AF800 QuantumDot (Qdot ®) Qdot ®525, Qdot ®565, Qdot ®585, Qdot ®605, based dyesQdot ®655, Qdot ®705, Qdot ®800 DyLight ™ Dyes (DL) DL549, DL649, DL680,DL800 Small fluorescing dyes FITC, Pacific Blue ™, Pacific Orange ™,Cascade Yellow ™, Marina blue ™, DSred, DSred-2, 7-AAD, TO- Pro-3,Cy-Dyes Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7 Phycobili Proteins:R-Phycoerythrin (RPE), PerCP, Allophycocyanin (APC), B-Phycoerythrin,C-Phycocyanin Fluorescent Proteins (E)GFP and GFP ((enhanced) greenfluorescent protein) derived mutant proteins; BFP, CFP, YFP, DsRed, T1,Dimer2, mRFP1, MBanana, mOrange, dTomato, tdTomato, mTangerine,mStrawberry, mCherry Tandem dyes with RPE RPE-Cy5, RPE-Cy5.5, RPE-Cy7,RPE-AlexaFluor ® tandem conjugates; RPE-Alexa610, RPE-TxRed Tandem dyeswith APC APC-Aleca600, APC-Alexa610, APC-Alexa750, APC- Cy5, APC-Cy5.5Calcium dyes Indo-1-Ca2+ Indo-2-Ca2+

Preferable labelling molecules employed in stationary cytometry and IHC:

-   -   Enzymatic labelling:        -   Horse radish peroxidase; reduces peroxides (H₂O₂), and the            signal is generated by the Oxygen acceptor when being            oxidized.            -   Precipitating dyes; Dyes that when they are reduced they                are soluble, and precipitate when oxidized, generating a                coloured deposit at the site of the reaction.            -   Precipitating agent, carrying a chemical residue, a                hapten, for second layer binding of binding molecules,                for amplification of the primary signal.            -   Luminol reaction, generating a light signal at the site                of reaction.        -   Other enzymes, such as Alkaline Phosphatase, capable of            converting a chemical compound from a non-detectable            molecule to a precipitated detectable molecule, which can be            coloured, or carries a hapten as described above.    -   Fluorescent labels; as those described for Flow cytometry are        likewise important for used in stationary cytometry, such as in        fluorescent microscopy.

Examples of preferable labels for stationary cytometry:

Enzyme substrate, Precipitate or Oxygen acceptor Residue, hapten* forBinding Chromogen/ secondary detection partner to Label precipitatingagent layer hapten HRP diaminobenzidine Colored precipitate — (DAB) HRP3-amino-9-ethyl- Colored precipitate — carbazole (AEC+) AP Fast red dyeRed precipitate — HRP biotinyl tyramide Exposed Biotin Streptavidin,residue avidine HRP fluorescein tyramide Exposed FluoresceinAnti-Fluorecein residue Antibody “Enzyme” Substrate that when Primarylabel; being a Secondary reacted precipitate dye, label in casechemiluminescence's, the primary or exposure of a label is a haptenhapten

In one embodiment the label comprises a connector molecule, whichconnector molecule is able to interact with a component on the linkerand/or binding molecule of the detection molecule. In one embodiment theconnector molecule is biotin or avidin. In one embodiment the linkercomprises streptavidin to which the label binds via its biotin or avidinconnector molecule.

Nucleic Acid Label

In one embodiment the detection molecule comprises at least one nucleicacid label, such as a nucleotide label, for example an oligonucleotidelabel.

In a preferred embodiment the label is an oligonucleotide. In apreferred embodiment, the label of the detection molecule is a DNAoligonucleotide (DNA label).

The terms nucleic acid label, nucleic acid molecule, nucleotide label,oligonucleotide label, DNA molecule, DNA label, DNA tag, DNAoligonucleotides and nucleic acid component may be used interchangeablyherein.

In one embodiment the nucleic acid label comprises one or more of thefollowing components:

-   -   barcode region,    -   5′ first primer region (forward)    -   3′ second primer region (reverse),    -   random nucleotide region,    -   connector molecule    -   stability-increasing components    -   short nucleotide linkers in between any of the above-mentioned        components    -   adaptors for sequencing    -   annealing region

Preferably the nucleic acid label comprises at least a barcode region(i.e. barcode sequence). A barcode region comprises a sequence ofconsecutive nucleic acids.

A nucleic acid label of the present invention comprises a number ofconsecutive nucleic acids. The nucleic acid can be any type of nucleicacid or modifications thereof, naturally occurring or synthetically made(artificial nucleic acids).

In one embodiment the nucleic acid label comprises or consists of DNA.

In another embodiment the nucleic acid label comprises or consists ofRNA.

In yet another embodiment the nucleic acid label comprises or consistsof artificial nucleic acids or Xeno nucleic acid (XNA).

Artificial nucleic acid analogs have been designed and synthesized bychemists, and include peptide nucleic acid (PNA), morpholino- and lockednucleic acid (LNA), as well as glycol nucleic acid (GNA), threosenucleic acid (TNA), HNA and CeNA. Each of these is distinguished fromnaturally occurring DNA or RNA by changes to the backbone of themolecule.

In yet another embodiment the nucleic acid label comprises or consistsof one or more of XNA, PNA, LNA, TNA, GNA, HNA and CeNA,

In a further embodiment the at least one nucleic acid molecule comprisesor consists of DNA, RNA, and/or artificial nucleotides such as PLA orLNA. Preferably DNA, but other nucleotides may be included to e.g.increase stability.

In a preferred embodiment the oligonucletide used in the invention is anatural oligonucleotide such as DNA or RNA, or it may be PNA, LNA, oranother type of unnatural oligonucleotide. The oligonucletides may bemodified on the base entity, the sugar entity, or in the linkerconnecting the individual nucleotides.

The length of the nucleic acid molecule may also vary. Thus, in oneembodiment the at least one nucleic acid molecule has a length in therange 20-100 nucleotides, such as 30-100, such as 30-80, such as 30-50nucleotides.

In one embodiment the label is an oligonucleotide of length 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31-35, 36-50, 51-100, or more than 100nucleotides.

In one embodiment the nucleic acid label comprises 1 to 1,000,000nucleic acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40 nucleic acids; for example 1-3, 3-5,5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-50, 50-60, 60-70,70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150,150-175, 175-200, 200-250, 250-300, 300-400, 400-500, 500-600, 600-700,700-800, 800-900, 900-1000, 1000-1500, 1500-2000, 2000-3000, 3000-4000,4000-5000, 5000-7500, 7500-10,000, 10,000-100,000, 100,000-1,000,000nucleic acids.

A nucleic acid label of the present invention as minimum comprises anumber of consecutive nucleic acids. The sequence of the nucleic acidsserves as a code that can be identified, such as amplified and/orsequenced.

The identifiable consecutive nucleic acids, or the identifiablesequence, of the nucleic acid label are denoted a ‘barcode’, ‘barcoderegion’, ‘nucleic acid barcode’, ‘unique sequence’, ‘unique nucleotidesequence’ and ‘coding sequence’ herein (used interchangably). Thebarcode region comprises of a number of consecutive nucleic acids makingup a nucleic acid sequence.

In one embodiment the nucleic acid label comprises a central stretch ofnucleic acids (barcode region) designed to be amplified by e.g. PCR.

In one embodiment, a nucleic acid barcode is a unique oligo-nucleotidesequence ranging for 10 to more than 50 nucleotides. In this embodiment,the barcode has shared amplification sequences in the 3′ and 5′ ends,and a unique sequence in the middle. This unique sequence can berevealed by sequencing and can serve as a specific barcode for a givenbinding molecule.

The unique sequence, the barcode, is composed of a series of nucleotidesthat together forms a sequence (series of nucleotides) that can bespecifically identified based on its composition. This sequencecomposition enables barcode #1 to be distinguishable from barcode #2,#3, #4 etc, up to more than 100.000 barcodes, based solely on the uniquesequence of each barcode. The complete nucleotide barcode may also becomposed of a combination of series of unique nucleotide sequenceslinked to each other. The series of unique sequences will togetherassign the barcode.

In one embodiment, each unique nucleotide sequence (barcode) holds 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31-35, 36-50, 51-100, or more than 100nucleotides (nucleic acids).

In a preferred embodiment the label is an oligonucleotide, where theunique sequence has a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31-35, 36-50, 51-100, or more than 100 nucleotides. In one embodimentthe unique sequence is shorter than the total length of the label.

In one embodiment the barcode region comprises or consists of 2-5, 5-10,10-15, 15-20, 20-25, 25-30, 30-40, 40-45, 45-50, 50-60, 60-70, 70-80,80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-175,175-200, 200-250, 250-300, 300-400, 400-500 nucleic acids.

The unique nucleotide sequence (barcode) is solely used as anidentification tag for the molecular interaction between the bindingmolecule and its target. The unique nucleotide sequences preferably arenot identical to any natural occurring DNA sequence, although sequencesimilarities or identities may occur.

Each nucleic acid barcode should hold sufficient difference from theadditional barcodes in a given experiment to allow specificidentification of a given barcode, distinguishable from the others.

The nucleic acid component (preferably DNA) has a special structure.Thus, in an embodiment the at least one nucleic acid molecule (label) iscomposed of at least a 5′ first primer region, a central region (barcoderegion), and a 3′ second primer region. In this way the central region(the barcode region) can be amplified by a primer set.

The coupling of the nucleic acid molecule to the backbone may also vary.Thus, in one embodiment the at least one nucleic acid molecule is linkedto said backbone via a streptavidin-biotin binding and/orstreptavidin-avidin binding. Other coupling moieties may also be used.

In one embodiment the nucleic acid label comprises a connector molecule,which connector molecule is able to interact with a component on thelinker and/or binding molecule of the detection molecule. In oneembodiment the connector molecule is biotin or avidin. In one embodimentthe linker comprises streptavidin to which the label binds via itsbiotin or avidin connector molecule.

In one embodiment the nucleic acid label comprises a random nucleotideregion. This random nt region is a potential tool for detecting labelcontaminants. A random nt region of the invention in one embodimentcomprises from 3-20 nucleotides, such as 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19 or 20 nt.

In some embodiments, the different labels used in an experiment possessthe same amplification properties and share common primer regions:Common primer regions together with shared amplification properties willensure that all labels that are present after cellular interaction andsorting are amplified equally whereby no sequences will be biased due tothe sequencing reaction.

With identical primer regions on differing labels there is an inherentrisk of contaminating one label with another—especially followingamplification reactions. To be able to trace potential contaminants ashort ‘random nucleotide region’ can be included in the nucleic acidlabel. Since the random nucleotide region is unique for each label, itwill be possible to inspect the sequencing data and see whether numerousreads of a given label is present. I.e. the random nucleotide region isa clonality control region. In one embodiment the random nucleotideregion consist of 2-20 nucleic acids; such as 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleic acids. A randomnucleotide region consisting of 6 nucleotides may be denoted ‘N6’herein, and so forth.

In one embodiment the nucleic acid label comprises one or morestability-increasing components (such as HEG or TEG)

The label is preferably stable when mixing with cells: as this mayexpose the label to nuclease digestion. A measure to minimize this maybe to add modifications in the form of hexaethylene glycol (HEG) or TEGat one or both ends of the oligonucleotide label.

Additionally stability can be accounted for in the buffers applied byadding constituents that exert a protective effect towards theoligo-nucleotides, e.g herring DNA and EDTA

In one embodiment the nucleic acid label comprises a sample identifyingsequence. To be able to analyze more than a single sample in eachsequencing reaction the nucleic acid labels may be appointed anadditional recognition feature, namely a sample identifying sequence.The sample identifying sequence is not a part of the initial design ofthe label, but will be appointed after cellular interaction and sortingvia primers in a PCR—thus all cells originating from the same sample,will have the same sample identification sequence. In one embodiment thesample identifying sequence is a short sequence, consisting of 2-20nucleic acids; such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 nucleic acids. The sample identifying sequence maybe attached to a primer, such as the forward primer.

The nucleic acid label is in one embodiment a ‘1 oligo system’comprising a forward primer, a barcode region and a reverse primer.

The nucleic acid label is in one embodiment a ‘2 oligo system’ with twosequences, the first comprising a forward primer, a barcode region and aannealing region; and the second comprising an annealing region, abarcode region and a reverse primer.

Peptide Label

In one embodiment the label, or the coding label, is a peptide labelcomprising a stretch of consecutive amino acid residues. This is the‘coding region’ the identity of which can be determined.

In one embodiment the peptide label comprises or consists of a definednumber of consecutive amino acids. It follows that the nucleic acidlabel in one embodiment comprises 2 or more consecutive amino acids,such as 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13,13-14, 14-15, 15-16, 16-17, 17-18, 18-19, 19-20, 20-21, 21-22, 22-23,23-24, 24-25, 25-26, 26-27, 27-28, 28-29, 29-30, 30-31, 31-32, 32-33,33-34, 34-35, 35-36, 36-37, 37-38, 38-39, 39-40, 40-45, 45-50, 50-55,55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 100-110,110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190,190-200, 200-225, 225-250, 250-275, 275-300, 300-350, 350-400, 400-450,450-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1500,1500-2000, or more than 2000, consecutive amino acids.

In one embodiment the peptide label comprises a stretch of consecutiveamino acid residues (coding region) and a protease cleavage site. Theprotease cleavage site is preferably located proximal to the linker thatconnects the label to the binding molecule.

When the detection molecule is brought into proximity of a protease, thepeptide label is cleaved and the coding region released from thedetection molecule. The sample cells may be precipitated and thesupernatant can be analysed by mass spectrometry to determine theidentity and amount of the labels that was released.

Proteases capable of cleaving the peptide labels may be coated on thesurface of sample cells, for example by adding antibody-proteaseconjugates where the antibody recognizes a particular cell surfacestructure.

In one embodiment the peptide label comprises natural (or standard)amino acids. In another embodiment the peptide label comprisesnon-naturally occurring amino acids (non-proteinogenic or non-standard).In one embodiment the peptide label comprises standard and non-standardamino acids.

A natural amino acid is a naturally occurring amino acid existing innature and being naturally incorporated into polypeptides(proteinogenic). They consist of the 20 genetically encoded amino acidsAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Tyr, Thr, Trp, Val, and 2 which are incorporated into proteinsby unique synthetic mechanisms: Sec (selenocysteine, or U) and Pyl(pyrrolysine, O). These are all L-stereoisomers.

Aside from the 22 natural or standard amino acids, there are many othernon-naturally occurring amino acids (non-proteinogenic or non-standard).They are either not found in proteins, or are not produced directly andin isolation by standard cellular machinery. Non-standard amino acidsare usually formed through modifications to standard amino acids, suchas post-translational modifications.

Any amino acids according to the present invention may be in the L- orD-configuration.

The standard and/or non-standard amino acids may be linked by peptidebonds to form a linear peptide chain.

The term peptide also embraces post-translational modificationsintroduced by chemical or enzyme-catalyzed reactions, as are known inthe art. Also, functional equivalents may comprise chemicalmodifications such as ubiquitination, labeling (e.g., withradionuclides, various enzymes, etc.), pegylation (derivatization withpolyethylene glycol), or by insertion (or substitution by chemicalsynthesis) of amino acids (amino acids) which do not normally occur inhuman proteins.

Protein post-translational modification (PTM) increases the functionaldiversity of the proteome by the covalent addition of functional groupsor proteins, proteolytic cleavage of regulatory subunits or degradationof entire proteins. These modifications include phosphorylation,glycosylation, ubiquitination, nitrosylation, methylation, acetylation,lipidation (C-terminal glycosyl phosphatidylinositol (GPI) anchor,N-terminal myristoylation, S-myristoylation, S-prenylation), amidation,and proteolysis and influence almost all aspects of normal cell biologyand pathogenesis.

Sterically similar compounds may be formulated to mimic the key portionsof the peptide structure and that such compounds may also be used in thesame manner as the peptides of the invention. This may be achieved bytechniques of modelling and chemical designing known to those of skillin the art. For example, esterification and other alkylations may beemployed to modify the amino terminus of e.g a di-arginine peptidebackbone, to mimic a tetra peptide structure. It will be understood thatall such sterically similar constructs fall within the scope of thepresent invention.

Peptides with N-terminal alkylations and C-terminal esterifications arealso encompassed within the present invention. Functional equivalentsalso comprise glycosylated and covalent or aggregative conjugates formedwith the same molecules, including dimers or unrelated chemicalmoieties. Such functional equivalents are prepared by linkage offunctionalities to groups which are found in fragment including at anyone or both of the N- and C-termini, by means known in the art.

Binding Molecules

A binding molecule is a molecule that specifically associates covalentlyor non-covalently with a structure belonging to or associated with anentity in a sample. The defined structure in sample bound by a bindingmolecule is also called the target structure or target of the bindingmolecule. A typical target for a binding molecule is a ‘markermolecule’, which marker molecule is specific for a given cell or celltype.

Example binding molecule include but is not limited to proteins,antibodies (monoclonal or polyclonal, derived from any species e.g. man,mouse, rat, rabbit, pig or camel, monkey or may be recombinantantibodies), antibody fragments, MHC multimers (including but notlimited to MHC dextramers, MHC tetramers, MHC Pentamers, cellsexpressing MHC molecules, MHC-peptide molecules covalently ornon-covalently attached to beads, streptactin or other moleculestructures), scaffold molecules, ligands, small organic molecules,nucleic acids (e.g. DNA, RNA, PNA), polysaccharides, other polymers,Aptamers including nucleic acid aptamers and peptide aptamers, affimers,beads, cells, living cells, dead cells, naturally occurring cells,genetic modified cells, hybridoma cells, gene transfected cells, celllike structures (e.g. liposomes, micelles), multicomponent complexescomprising 2, 3, 4, 5, 6, 7, 8, or more binding molecule subunits, andsupramolecular structures, or other molecules, cells or substances ableto bind defined structure in sample.

A binding molecule is useful for detection of a given defined structurein sample if the binding molecule binds the defined structure with acertain affinity. In order to be specific the binding molecule has tohave a binding affinity that is higher than the binding moleculesbinding affinity for other structures in sample.

When the detection molecule comprises two or more binding molecules, thebinding molecule may be referred to as a binding molecule multimer. Whenthe detection molecule comprises two or more binding molecules, whereinthe binding molecules are MHC molecules or MHC complexes, the MHCcomplexes may be referred to as a MHC multimer.

Typical defined structures or targets to which the binding moleculeassociate include but is not limited to: surface receptors on cells(e.g. TCR, CD molecules, growth receptors, MHC complexes, mannosebinding receptor, transporter proteins), other structures on the surfaceof cells (e.g. lipids, sugars, proteins), intracellular substances incells (e.g. DNA, RNA, ribosomes, organelles, cytokines, transcriptionfactors, cytoskeleton components, intracellular proteins, sugars),components in fluidics (e.g. antibodies, blood plates, serum proteins,sugars), structures in interstitial space in tissues etc.

A binding molecule may have a molecular weight of between 50 Da andseveral million Da. In some instances a very low molecular weight ispreferred, such as a molecular weight of 50-250 Da, or 251-500 Da. Inother cases a low molecular weight, e.g. 501-2000 Da, 2001-5000 Da, or5001-10000 Da may be preferred. In yet other cases, a high molecularweight of the binding molecule is practical, and the molecular weight ofthe binding molecule may be 10001-50000 Da, 50001-200000 Da, or200000-1000000 Da. Finally, multi-molecule structures, such as in caseswhere a number of different fluorescent proteins are ordered in an arrayby binding to specific regions in a template DNA, where the totalbinding molecule thus is 50000-200000 Da, 200001-100000, or1000001-10000000 Da.

Examples of binding molecules and corresponding target molecules aregiven below:

-   -   I. Antibodies binding to membrane components on, or within        cells; e.g. Polysaccharides, proteins, or lipid residues.    -   II. MHC multimers (e.g. MHC dextramers, MHC tetramers, MHC        pentamers), optionally complexed with a specific peptide, binds        to the T-cell receptor (TCR) on T-cells.    -   III. MHC multimers (e.g. MHC dextramers, MHC tetramers, MHC        pentamers), complexed with a so-called nonsense peptide (i.e. a        peptide that binds the MHC protein but expectably does not        mediate efficient MHC complex-TCR interaction with any T-cell),        are used as negative control for the specific binding of a        specific MHC multimers to the cell.    -   IV. Binding molecules such as Propidium Iodide (PI) that stain        DNA in cells, here the binding molecule and the label can        optionally be the same molecule.    -   V. Binding molecules that stain DNA, and that is used to        characterize the state of a cell (e.g. state of cell cycle),        e.g. Draq 5, PI, or other DNA binding molecules.    -   VI. Binding molecules that bind specifically to incorporated        molecules. An example is BrdU, which may be added to a cell        which will incorporate BrdU into its DNA; by using anti-BrdU        antibody, cells that have incorporated BrdU will be detected.    -   VII. Binding molecules such as hormones, or growth factors, that        specifically interacts with a cellular component, such as the        estrogen receptor with estrogen or the EGF receptor with EGF.    -   VIII. Introduction of modified nucleotides, amino acids, or        vitamins into cells which incorporate these into cellular        components that subsequently can be detected, by themselves        (e.g. if they are radioactive, or fluoresce) or by their        association with a detection molecule.

In one embodiment the binding molecule is an MHC or MHC like complex,such as CD1a, CD1b, CD1c, or CD1d in complex with a peptide, lipid,glycolipid or any other molecule that binds to CD1a, CD1b, CD1c, orCD1d, or such as MR1 in complex with any epitope that binds to the MR1complex, or such as an empty CD1a, CD1b, CD1c, CD1d or MR1, i.e. withoutepitope bound.

In one embodiment the detection molecule is CD1, such as CD1 selectedfrom the group consisting of CD1 CD1a, CD1b, CD1c, CD1d and CD1e.

CD1 (cluster of differentiation 1) is a family of glycoproteinsexpressed on the surface of various human antigen-presenting cells. Theyare related to the class I MHC molecules, and are involved in thepresentation of lipid antigens to T cells.

In one embodiment the detection molecule is a MHC Class I-like proteins;such as MIC A, MIC B, CD1d, HLA E, HLA F, HLA G, HLA H, ULBP-1, ULBP-2,and ULBP-3.

In one embodiment the binding molecule of the invention comprises onepeptide-major histocompatibility complex (pMHC) or MHC complex. Inanother embodiment the binding molecule of the invention comprises 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, such as1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13,13-15, 15-16, 16-20, 20-21, 21-50, 50-100, 51-100, or more than 100 MHCcomplexes, such as peptide-MHC complexes.

In a particular embodiment the binding molecule of the detectionmolecule is an oligonucleotide, wherein said oligonucleotide preferablybinds to DNA or RNA molecules inside a cell. Optionally, the identity ofthe detection molecule within a cell is determined by extending theoligonucleotide-binding molecule using a polymerase, in this wayidentifying the detection molecule as well as the RNA or DNA template towhich it is attached.

The MHC multimer can be composed of MHC class I, class II, CD1 or otherMHC-like molecules. Thus, when the term MHC multimers is used hereinthis includes all MHC-like molecules. The MHC multimer is formed throughmultimerization of peptide-MHC molecules via different backbones.

An aspect of the invention relates to a multimeric majorhistocompatibility complex (MHC) comprising

-   -   two or more MHC's linked by a backbone molecule; and    -   at least one nucleic acid molecule linked to said backbone,        wherein said nucleic acid molecule comprises a central stretch        of nucleic acids (barcode region) designed to be amplified by        e.g. PCR.

Different types of backbones may be used. Thus, in an embodiment thebackbone molecule is selected from the group consisting ofpolysaccharides, such as glucans such as dextran, a streptavidin or astreptavidin multimer. The skilled artisan may find other alternativebackbones.

The MHC's may be coupled to the backbone by different means. Thus, in anembodiment the MHC's are coupled to the backbone through astreptavidin-biotin binding or a streptavidin-avidin binding. Againother binding moieties may be used. The specific binding may usespecific couplings points. In another embodiment the MHC's are linked tothe backbone via the MHC heavy chain.

The MHC consists of different elements, which may partly be expressedand purified from cell systems (such as the MHC heavy chain and theBeta-2-microglobulin element). Alternatively, the elements may bechemically synthesized. The specific peptide is preferably chemicallysynthesized.

All three elements are required for the generation of a stable MHC(complex). Thus, in an embodiment the MHC is artificially assembled.

The multimeric MHC may comprise different numbers of MHC's. Thus, in yetan embodiment the multimeric major histocompatibility complex (MHC) iscomposed of at least four MHC's, such as at least eight, such as atleast ten, 2-30, 2-20, such as 2-10 or such as 4-10 MHC's.

In one embodiment there is provided a detection molecule comprising twoor more MHC complexes and a multimerization domain, such as comprising 2MHC complexes, such as 3 MHC complexes, for example 4 MHC complexes,such as 5 MHC complexes, for example 6 MHC complexes, such as 7 MHCcomplexes, for example 8 MHC complexes, such as 9 MHC complexes, forexample 10 MHC complexes, such as 11 MHC complexes, for example 12 MHCcomplexes, such as 13 MHC complexes, for example 14 MHC complexes, suchas 15 MHC complexes, for example 16 MHC complexes, such as 17 MHCcomplexes, for example 18 MHC complexes, such as 19 MHC complexes, forexample 20 MHC complexes, such as 21 MHC complexes, for example 22 MHCcomplexes, such as 23 MHC complexes, for example 24 MHC complexes, suchas 25 MHC complexes, for example 26 MHC complexes, such as 27 MHCcomplexes, for example 28 MHC complexes, such as 29 MHC complexes, forexample 30 MHC complexes, such as more than 30 MHC complexes.

In one embodiment there is provided a detection molecule comprising twoor more MHC complexes and a multimerization domain, such as 2-3 MHCcomplexes, for example 3-4 MHC complexes, such as 4-5 MHC complexes, forexample 5-6 MHC complexes, such as 6-7 MHC complexes, for example 7-8MHC complexes, such as 8-9 MHC complexes, for example 9-10 MHCcomplexes, such as 10-11 MHC complexes, for example 11-12 MHC complexes,such as 12-13 MHC complexes, for example 13-14 MHC complexes, such as14-15 MHC complexes, for example 15-16 MHC complexes, such as 16-17 MHCcomplexes, for example 17-18 MHC complexes, such as 18-19 MHC complexes,for example 19-20 MHC complexes, such as 20-25 MHC complexes, forexample 25-30 MHC complexes, such as 30-35 MHC complexes, for example35-40 MHC complexes, such as 40-45 MHC complexes, for example 45-50 MHCcomplexes, such as 50-55 MHC complexes, for example 55-60 MHC complexes,such as 60-65 MHC complexes, for example 65-70 MHC complexes, such as70-75 MHC complexes, for example 75-80 MHC complexes, such as 80-85 MHCcomplexes, for example 85-90 MHC complexes, such as 90-95 MHC complexes,for example 95-100 MHC complexes, such as 100-125 MHC complexes, forexample 125-150 MHC complexes, such as 150-175 MHC complexes, forexample 175-200 MHC complexes, such as more than 200 MHC complexes.

In one embodiment the detection molecule comprises 2-20 MHC complexes,for example 2-10 MHC complexes, such as 4-20 MHC complexes, for example4-10 MHC complexes, such as 5-10 MHC complexes, for example 5-20 MHCcomplexes, such as 5-25 MHC complexes.

In one embodiment there is provided a detection molecule comprising twoor more MHC complexes and a multimerization domain, and comprising oneor more labels, such as DNA labels, as defined herein elsewhere.

Different types of MHC's may form part of the multimer. Thus, in anembodiment the MHC is selected from the group consisting of class I MHC,a class II MHC, a CD1, or a MHC-like molecule. For MHC class I thepresenting peptide is a 9-11 mer peptide; for MHC class II, thepresenting peptide is 12-18 mer peptides. For alternative MHC-moleculesit may be fragments from lipids or gluco-molecules which are presented.

In one embodiment the binding molecule comprises a connector molecule,which connector molecule is able to interact with a component on thelinker and/or label of the detection molecule. In one embodiment theconnector molecule is biotin or avidin. In one embodiment the linkercomprises streptavidin to which the binding molecule binds via itsbiotin or avidin connector molecule.

BM: MHC Complexes

In a preferred embodiment the binding molecule of the detection moleculeof the present invention comprises one or more MHC complexes (or MHCmolecules).

The present invention in one embodiment provides a detection moleculecomprising a binding molecule (BM), a linker (Li) and a label (La),wherein the binding molecule comprises one or more MHC complexes.

The present invention in one embodiment provides a detection moleculecomprising a binding molecule (BM), a linker (Li) and a label (La),wherein the binding molecule comprises one or more MHC complexes, andthe label is a nucleic acid label, such as a DNA label.

The present invention in one embodiment provides a detection moleculecomprising a binding molecule (BM), a linker (Li) and a label (La),wherein the binding molecule comprises one or more MHC complexes, thelabel is a nucleic acid label, such as a DNA label, and the linker is amultimerization domain (or carrier molecule, or backbone).

The present invention in one embodiment provides a detection moleculecomprising a binding molecule (BM), a linker (Li) and a label (La),wherein the binding molecule comprises two or more MHC complexes, thelinker is a dextran multimerization domain (also known as ‘MHCdextramers’), and the label is a nucleic acid label, such as a DNAlabel.

The MHC as a binding molecule (MHC-BM) according to the presentinvention is described in more detail herein below. The MHC as a bindingmolecule according to the present invention in combination with somelinkers is also disclosed in detail herein below.

Each embodiment wherein the binding molecule is a MHC complex can becombined individually with any of the herein disclosed furthercomponents of the detection molecules namely linker and label.

The present invention in one aspect refers to a detection moleculecomprising a MHC monomer comprising a-b-P, or a MHC multimer comprising(a-b-P)_(n), wherein n>1,

wherein a and b together form a functional MHC protein capable ofbinding an antigenic peptide P,

wherein (a-b-P) is a MHC-peptide complex formed when the antigenicpeptide P binds to the functional MHC protein.

The present invention in another aspect refers to a MHC monomercomprising a-b-P, or a MHC multimer comprising (a-b-P)_(n), wherein n>1,

wherein a and b together form a functional MHC protein capable ofbinding an antigenic peptide P,

wherein (a-b-P) is a MHC-peptide complex formed when the antigenicpeptide P binds to the functional MHC protein, and

wherein each MHC complex or MHC peptide complex of a MHC multimer isassociated with one or more multimerization domains.

MHC monomers and MHC multimers comprising two or more MHC complexesand/or MHC peptide complexes of class 1 or class 2 MHC are covered bythe present invention.

In another aspect the present invention is directed to a compositioncomprising a plurality of MHC monomers and/or MHC multimers according tothe present invention, wherein the MHCs of the MHC multimersindividually are identical or different, and a linker such as a carrier.

The present invention further relates to a method for detection ofantigen-specific T cells, said method comprising the steps of 1)providing a detection molecule comprising a MHC multimer, 2) providing apopulation of antigen-specific T cells, and 3) detectingantigen-specific T cells specific for the peptide P of the MHC multimer.

In a further embodiment the present invention relates to a method forcounting of antigen-specific T cells, said method comprising the stepsof 1) providing a detection molecule comprising a MHC multimer, 2)providing a population of antigen-specific T cells, and 3) countingantigen-specific T cells specific for the peptide P of the MHC multimer.

The present invention also relates to a method for sorting ofantigen-specific T cells, said method comprising the steps of 1)providing a detection molecule comprising a MHC multimer, 2) providing apopulation of antigen-specific T cells, and 3) sorting antigen-specificT cells specific for the peptide P of the MHC multimer.

In yet another embodiment the present invention relates to a method forisolation of antigen-specific T cells, said method comprising the stepsof 1) providing a detection molecule comprising a MHC multimer, 2)providing a population of antigen-specific T cells, and 3) isolatingantigen-specific T cells specific for the peptide P of the MHC multimer.

In a still further aspect there is provided a method for immunemonitoring of one or more diseases comprising monitoring ofantigen-specific T cells, said method comprising the steps of

-   -   i) providing a detection molecule comprising a MHC monomer or        MHC multimer or individual components thereof according to the        present invention,    -   ii) providing a population of antigen-specific T cells or        individual antigen-specific T cells, and    -   iii) measuring the number, activity or state and/or presence of        antigen-specific of T cells specific for the peptide P of the        said MHC monomer or MHC multimer, thereby immune monitoring said        one or more diseases.

In one aspect, the present invention is directed to MHC complexescomprising a linker which is a multimerization domain, preferablycomprising a carrier molecule and/or a scaffold.

There is also provided a MHC multimer comprising 2 or more MHC-peptidecomplexes and a multimerization domain to which the 2 or moreMHC-peptide complexes are associated. The MHC multimer can generally beformed by association of the 2 or more MHC complexes with themultimerization domain to which the 2 or more MHC complexes are capableof associating.

The multimerization domain can be a scaffold associated with one or moreMHC complexes, or a carrier associated with one or more, preferably morethan one, MHC complex(es), or a carrier associated with a plurality ofscaffolds each associated with one or more MHC complexes, such as 2 MHCcomplexes, 3 MHC complexes, 4 MHC complexes, 5 MHC complexes or morethan 5 MHC complexes. Accordingly, multimerization domain collectivelyrefers to each and every of the above. It will be clear from thedetailed description of the invention provided herein below when themultimerization domain refers to a scaffold or a carrier or a carriercomprising one or more scaffolds. It is understood that MHC complexes ofthe invention may or may not be loaded with antigenic peptides; whenthey are this may be referred to as a MHC-peptide complex.

Generally, when a multimerization domain comprising a carrier and/or ascaffold is present, the MHC complexes can be associated with thisdomain either directly or via one or more binding entities. Theassociation can be covalent or non-covalent.

Accordingly, there is provided in one embodiment a MHC complexcomprising one or more entities (a-b-P)_(n), wherein a and b togetherform a functional MHC protein capable of binding a peptide P, andwherein (a-b-P) is the MHC-peptide complex formed when the peptide Pbinds to the functional MHC protein, said MHC complex optionally furthercomprising a multimerization domain comprising a carrier molecule and/ora scaffold.

“MHC complex” refers to any MHC complex, including MHC monomers in theform of a single MHC complex and MHC multimers comprising amultimerization domain to which more than one MHC complex is associated.MHC complexes can be with or without peptide P included in the bindinggroove of the MHC.

When the invention is directed to complexes comprising a MHC multimer,i.e. a plurality of MHC complexes of the general composition (a-b-P)_(n)associated with a multimerization domain, n is by definition more than1, i.e. at least 2 or more. Accordingly, the term “MHC multimer” is usedherein specifically to indicate that more than one MHC complex isassociated with a multimerization domain, such as a scaffold or carrieror carrier comprising one or more scaffolds. Accordingly, a single MHCcomplex can be associated with a scaffold or a carrier or a carriercomprising a scaffold and a MHC-multimer comprising 2 or more MHCcomplexes can be formed by association of the individual MHC complexeswith a scaffold or a carrier or a carrier comprising one or morescaffolds each associated with one or more MHC complexes.

When the MHC complex comprises a multimerization domain to which the nMHC complexes are associated, the association can be a covalent linkageso that each or at least some of the n MHC complexes is covalentlylinked to the multimerization domain, or the association can be anon-covalent association so that each or at least some of the n MHCcomplexes are non-covalently associated with the multimerization domain.

The MHC complexes of the invention may be provided in non-soluble orsoluble form, depending on the intended application.

Effective methods to produce a variety of MHC complexes comprisinghighly polymorphic human HLA encoded proteins makes it possible toperform advanced analyses of complex immune responses, which maycomprise a variety of peptide epitope specific T-cell clones.

One of the benefits of the MHC complexes of the present invention isthat the MHC complexes overcome low intrinsic affinities of monomerligands and counter receptors. The MHC complexes have a large variety ofapplications that include targeting of high affinity receptors (e.g.hormone peptide receptors for insulin) on target cells. Taken togetherpoly-ligand binding to target cells has numerous practical, clinical andscientifically uses.

Thus, the present invention provides MHC complexes which presentmono-valent or multi-valent binding sites for MHC-peptide recognisingcells, such as MHC complexes optionally comprising a multimerizationdomain, such as a scaffold or a carrier molecule, which multimerizationdomain have attached thereto, directly or indirectly via one or moreconnectors, covalently or non-covalently, one or more MHC complexes.“One or more” as used herein is intended to include one as well as aplurality, such as at least 2. This applies i.a. to the MHC complexesand to the binding entities of the multimerization domain. The scaffoldor carrier molecule may thus have attached thereto a MHC complex or aplurality of such MHC complexes, and/or a connector or a plurality ofconnectors.

In one preferred embodiment the MHC multimer is between 50,000 Da and1,000,000 Da, such as from 50,000 Da to 980,000; for example from 50,000Da to 960,000; such as from 50,000 Da to 940,000; for example from50,000 Da to 920,000; such as from 50,000 Da to 900,000; for examplefrom 50,000 Da to 880,000; such as from 50,000 Da to 860,000; forexample from 50,000 Da to 840,000; such as from 50,000 Da to 820,000;for example from 50,000 Da to 800,000; such as from 50,000 Da to780,000; for example from 50,000 Da to 760,000; such as from 50,000 Dato 740,000; for example from 50,000 Da to 720,000; such as from 50,000Da to 700,000; for example from 50,000 Da to 680,000; such as from50,000 Da to 660,000; for example from 50,000 Da to 640,000; such asfrom 50,000 Da to 620,000; for example from 50,000 Da to 600,000; suchas from 50,000 Da to 580,000; for example from 50,000 Da to 560,000;such as from 50,000 Da to 540,000; for example from 50,000 Da to520,000; such as from 50,000 Da to 500,000; for example from 50,000 Dato 480,000; such as from 50,000 Da to 460,000; for example from 50,000Da to 440,000; such as from 50,000 Da to 420,000; for example from50,000 Da to 400,000; such as from 50,000 Da to 380,000; for examplefrom 50,000 Da to 360,000; such as from 50,000 Da to 340,000; forexample from 50,000 Da to 320,000; such as from 50,000 Da to 300,000;for example from 50,000 Da to 280,000; such as from 50,000 Da to260,000; for example from 50,000 Da to 240,000; such as from 50,000 Dato 220,000; for example from 50,000 Da to 200,000; such as from 50,000Da to 180,000; for example from 50,000 Da to 160,000; such as from50,000 Da to 140,000; for example from 50,000 Da to 120,000; such asfrom 50,000 Da to 100,000; for example from 50,000 Da to 80,000; such asfrom 50,000 Da to 60,000; such as from 100,000 Da to 980,000; forexample from 100,000 Da to 960,000; such as from 100,000 Da to 940,000;for example from 100,000 Da to 920,000; such as from 100,000 Da to900,000; for example from 100,000 Da to 880,000; such as from 100,000 Dato 860,000; for example from 100,000 Da to 840,000; such as from 100,000Da to 820,000; for example from 100,000 Da to 800,000; such as from100,000 Da to 780,000; for example from 100,000 Da to 760,000; such asfrom 100,000 Da to 740,000; for example from 100,000 Da to 720,000; suchas from 100,000 Da to 700,000; for example from 100,000 Da to 680,000;such as from 100,000 Da to 660,000; for example from 100,000 Da to640,000; such as from 100,000 Da to 620,000; for example from 100,000 Dato 600,000; such as from 100,000 Da to 580,000; for example from 100,000Da to 560,000; such as from 100,000 Da to 540,000; for example from100,000 Da to 520,000; such as from 100,000 Da to 500,000; for examplefrom 100,000 Da to 480,000; such as from 100,000 Da to 460,000; forexample from 100,000 Da to 440,000; such as from 100,000 Da to 420,000;for example from 100,000 Da to 400,000; such as from 100,000 Da to380,000; for example from 100,000 Da to 360,000; such as from 100,000 Dato 340,000; for example from 100,000 Da to 320,000; such as from 100,000Da to 300,000; for example from 100,000 Da to 280,000; such as from100,000 Da to 260,000; for example from 100,000 Da to 240,000; such asfrom 100,000 Da to 220,000; for example from 100,000 Da to 200,000; suchas from 100,000 Da to 180,000; for example from 100,000 Da to 160,000;such as from 100,000 Da to 140,000; for example from 100,000 Da to120,000; such as from 150,000 Da to 980,000; for example from 150,000 Dato 960,000; such as from 150,000 Da to 940,000; for example from 150,000Da to 920,000; such as from 150,000 Da to 900,000; for example from150,000 Da to 880,000; such as from 150,000 Da to 860,000; for examplefrom 150,000 Da to 840,000; such as from 150,000 Da to 820,000; forexample from 150,000 Da to 800,000; such as from 150,000 Da to 780,000;for example from 150,000 Da to 760,000; such as from 150,000 Da to740,000; for example from 150,000 Da to 720,000; such as from 150,000 Dato 700,000; for example from 150,000 Da to 680,000; such as from 150,000Da to 660,000; for example from 150,000 Da to 640,000; such as from150,000 Da to 620,000; for example from 150,000 Da to 600,000; such asfrom 150,000 Da to 580,000; for example from 150,000 Da to 560,000; suchas from 150,000 Da to 540,000; for example from 150,000 Da to 520,000;such as from 150,000 Da to 500,000; for example from 150,000 Da to480,000; such as from 150,000 Da to 460,000; for example from 150,000 Dato 440,000; such as from 150,000 Da to 420,000; for example from 150,000Da to 400,000; such as from 150,000 Da to 380,000; for example from150,000 Da to 360,000; such as from 150,000 Da to 340,000; for examplefrom 150,000 Da to 320,000; such as from 150,000 Da to 300,000; forexample from 150,000 Da to 280,000; such as from 150,000 Da to 260,000;for example from 150,000 Da to 240,000; such as from 150,000 Da to220,000; for example from 150,000 Da to 200,000; such as from 150,000 Dato 180,000; for example from 150,000 Da to 160,000.

In another preferred embodiment the MHC multimer is between 1,000,000 Daand 3,000,000 Da, such as from 1,000,000 Da to 2,800,000; for examplefrom 1,000,000 Da to 2,600,000; such as from 1,000,000 Da to 2,400,000;for example from 1,000,000 Da to 2,200,000; such as from 1,000,000 Da to2,000,000; for example from 1,000,000 Da to 1,800,000; such as from1,000,000 Da to 1,600,000; for example from 1,000,000 Da to 1,400,000.

Number of MHC Complexes Per Multimer

A non-exhaustive list of possible MHC mono- and multimers illustratesthe possibilities. ‘n’ indicates the number of MHC complexes comprisedin the multimer:

-   a) n=1, Monomers-   b) n=2, Dimers, multimerization can be based on IgG scaffold,    streptavidin with two MHC's, coiled-coil dimerization e.g. Fos.Jun    dimerization-   c) n=3, Trimers, multimerization can be based on streptavidin as    scaffold with three MHC's, TNFalpha-MHC hybrids, triplex DNA-MHC    conjugates or other trimer structures-   d) n=4, Tetramers, multimerization can be based on streptavidin with    all four binding sites occupied by MHC molecules or based on dimeric    IgA-   e) n=5, Pentamers, multimerization can take place around a    pentameric coil-coil structure-   f) n=6, Hexamers-   g) n=7, Heptamers-   h) n=8-12, Octa-dodecamers, multimerization can take place using    Streptactin-   i) n=10, Decamers, multimerization can take place using IgM-   j) 1<n<100, Dextramers, as multimerization domain polymers such as    polypeptide, polysaccharides and Dextrans can be used.-   k) 1<n<1000, Multimerization can make use of dendritic cells (DC),    antigen-presenting cells (APC), micelles, liposomes, beads, surfaces    e.g. microtiter plate, tubes, microarray devices, micro-fluidic    systems-   l) 1<n, n in billions or trillions or higher, multimerization take    place on beads, and surfaces e.g. microtiter plate, tubes,    microarray devices, micro-fluidic systems

MHC multimers thus include MHC-dimers, MHC-trimers, MHC-tetramers,MHC-pentamers, MHC-hexamers, as well as organic molecules, cells,membranes, polymers and particles that comprise two or more MHC-peptidecomplexes. Example organic molecule-based multimers includefunctionalized cyclic structures such as benzene rings where e.g. abenzene ring is functionalized and covalently linked to e.g. three MHCcomplexes; example cell-based MHC multimers include dendritic cells andantigen presenting cells (APCs); example membrane-based MHC multimersinclude liposomes and micelles carrying MHC-peptide complexes in theirmembranes; example polymer-based MHC multimers include MHC-dextramers(dextran to which a number of MHC-peptide complexes are covalently ornon-covalently attached) and example particles include beads or othersolid supports with MHC complexes immobilized on the surface. Obviously,any kind of multimerization domain can be used, including any kind ofcell, polymer, protein or other molecular structure, or particles andsolid supports.

More than 600 MHC alleles (class 1 and 2) are known in humans; for manyof these, the peptide binding characteristics are known. The frequencyof the different HLA alleles varies considerably, also between differentethnic groups. Thus it is of outmost importance to carefully select theMHC alleles that corresponds to the population that one wish to study.

Any of the components of a MHC complex can be of any of the belowmentioned origins. The list is non-exhaustive. A complete list wouldencompass all Chordate species. By origin is meant that the sequence isidentical or highly homologous to a naturally occurring sequence of thespecific species.

List of origins: Human, Mouse, Primate (Chimpansee, Gorilla, OrangUtan), Monkey (Macaques), Porcine (Swine/Pig), Bovine(Cattle/Antilopes), Equine (Horse), Camelides (Camels), Ruminants(Deears), Canine (Dog), Feline (Cat), Bird (Chicken, Turkey), Fish,Reptiles, Amphibians.

In one embodiment the binding molecule is a MHC class I complex ofHLA-type A.

In one embodiment the binding molecule is a MHC class I complex ofHLA-type B.

In one embodiment the binding molecule is a MHC class I complex ofHLA-type C.

In one embodiment the binding molecules is a MHC class I complex, ofsupertype HLA-A1 (eg. HLA-A*0101, HLA-A*2601, HLA-A*2602, HLA-A2603,HLA-A*3002, HLA-A*3003, HLA-A*3004, HLA-A*3201).

In one embodiment the binding molecules is a MHC class I complex, ofsupertype HLA-A01 A03 (eg. HLA-A*3001).

In one embodiment the binding molecules is a MHC class I complex, ofsupertype HLA-A01 A024 (eg. HLA-A*2902).

In one embodiment the binding molecules is a MHC class I complex, ofsupertype HLA-A2 (eg. HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204,HLA-A*0205, HLA-A*0206, HLA-A*0207, HLA-A*0214, HLA-A*0217, HLA-A*6802,HLA-A*6901).

In one embodiment the binding molecules is a MHC class I complex, ofsupertype HLA-A3 (eg. HLA-A*0301, HLA-A*1101, HLA-A*3101, HLA-A*3301,HLA-A*3303, HLA-A*6601, HLA-A*6801, HLA-A*7401).

In one embodiment the binding molecules is a MHC class I complex, ofsupertype HLA-A24 (eg. HLA-A*2301, HLA-A*2402).

In one embodiment the binding molecules is a MHC class I complex, ofsupertype HLA-B7 (eg. HLA-B*0702, HLA-B*0703, HLA-B*0705, HLA B*1508,HLA-B*3501, HLA-B*3503, HLA-B*4201, HLA-B*5101, HLA-B*5102, HLA-B*5103,HLA-B*5301, HLA-B*5401, HLA-B*5501, HLA-B*5502, HLA-B*5601, HLA-B*6701,HLA-B*7801).

In one embodiment the binding molecules is a MHC class I complex, ofsupertype HLA-B8 (eg. HLA-B*0801, HLA-B*0802).

In one embodiment the binding molecules is a MHC class I complex, ofsupertype HLA-B27 (eg. HLA-B*1402, HLA-B*1503, HLA-B*1509, HLA-B*1510,HLA-B*1518, HLA-B*2702, HLA-B*2703, HLA-B*2704, HLA-B*2705, HLA-B*2706,HLA-B*2707, HLA-B*2708, HLA-B*2709, HLA-B*3801, HLA-B*3901, HLA-B*3902,HLA-B*3909, HLA-B*4801, HLA-B*7301).

In one embodiment the binding molecules is a MHC class I complex, ofsupertype HLA-B44 (eg. HLA-B*1801, HLA-B*3701, HLA-B*4001, HLA-B*4002,HLA-B*4006, HLA-B*4402, HLA-B*4403, HLA-B*4501).

In one embodiment the binding molecules is a MHC class I complex, ofsupertype HLA-B58 (eg. HLA-B*1516, HLA-B*1517, HLA-B*5701, HLA-B*5702,HLA-B*5801, HLA-B*5802).

In one embodiment the binding molecules is a MHC class I complex, ofsupertype HLA-B62 (eg. HLA-B*1501, HLA-B*1502, HLA-B*1512, HLA-B*1513,HLA-B*4601, HLA-B*5201).

In one embodiment the binding molecules is a MHC class I complex, ofsupertype HLA Cw 1-8 (eg. HLA-C*01, HLA-C*02, HLA-C*03, HLA-C*04,HLA-C*05, HLA-C*06, HLA-C*07, HLA-C*08).

In one embodiment the binding molecule is a MHC class I complex, whichbinds peptides with an acidic amino acid on 3^(rd) position (eg.HLA-A*0101, HLA-A*2601, HLA-A*2602, HLA-A*2603, HLA-A*3002, HLA-A*3003,HLA-A*3004, HLA-A*3201).

In one embodiment the binding molecule is a MHC class I complex, whichbinds peptides with a hydrophobic amino acid on 9^(th) position (eg.HLA-A*0201 . . . 0207, A*0214, A*0217, A*6802, A*6901, HLA-B*1516,B*1517, B*5701, B*5702, B*5801, B*5802).

In one embodiment the binding molecule is a MHC class I complex, whichbinds peptides with a Basic amino acid on 9^(th) position (eg.HLA-A*0301, HLA-A*1101, HLA-A*3101, HLA-A*3301, HLA-A*3303, HLA-A*6601,HLA-A*6801, HLA-A*7401).

In one embodiment the binding molecule is a MHC class I complex, whichbinds peptides with a Tyrosine amino acid on 2^(nd) position (eg.HLA-A*2301, HLA-A*2402).

In one embodiment the binding molecule is a MHC class I complex, whichbinds peptides with a Proline amino acid on 2^(nd) position (eg.HLA-B*0702, HLA-B*0703, HLA-B*0705, HLA-B*1508, HLA-B*3501, HLA-B*3503,HLA-B*4201, HLA-B*5101, HLA-B*5102, HLA-B*5103, HLA-B*5301, HLA-B*5401,HLA-B*5501, HLA-B*5502, HLA-B*5601, HLA-B*6701, HLA-B*7801).

In one embodiment the binding molecule is a MHC class I complex, whichbinds peptides with a Lysine amino acid on 3^(rd) and 5^(th) position(eg. HLA-B*0801, B*0802).

In one embodiment the binding molecule is a MHC class I complex, whichbinds peptides with a Arginine amino acid on 2^(nd) position (eg.HLA-B*1402, HLA-B*1503, HLA-B*1509, HLA-B*1510, HLA-B*1518, HLA-B*2702,HLA-B*2703, HLA-B*2704, HLA-B*2705, HLA-B*2706, HLA-B*2707, HLA-B*2708,HLA-B*2709, HLA-B*3801, HLA-B*3901, HLA-B*3902, HLA-B*3909, HLA-B*4801,HLA-B*7301).

In one embodiment the binding molecule is a MHC class I complex, whichbinds peptides with a Glutamic acid amino acid on 2^(nd) position (eg.HLA-B*1801, HLA-B*3701, HLA-B*4001, HLA-B*4002, HLA-B*4006, HLA-B*4402,HLA-B*4403, HLA-B*4501).

In one embodiment the binding molecule is a MHC class I complex, whichbinds peptides with a Tyrosine amino acid on 9^(th) position (eg.HLA-B*1501, HLA-B*1502, HLA-B*1512, HLA-B*1513, HLA-B*4601, HLA-B*5201).

In one embodiment the binding molecule is a MHC class I complex, whichin the B pocket selectively binds small or aliphatic peptides (eg.HLA-A*0101, HLA-A*2601, HLA-A*2602, HLA-A*2603, HLA-A*3002, HLA-A*3003,HLA-A*3004, HLA-A*3201, HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204,HLA-A*0205, HLA-A*0206, HLA-A*0207, HLA-A*0214, HLA-A*0217, HLA-A*6802,HLA-A*6901, HLA-A*0301, HLA-A*1101, HLA-A*3101, HLA-A*3301, HLA-A*3303,HLA-A*6601, HLA-A*6801, HLA-A*7401).

In one embodiment the binding molecule is a MHC class I complex, whichin the F pocket selectively binds aliphatic peptides (eg. HLA-A*0101,HLA-A*2601 . . . 2603, HLA-A*3002, HLA-A*3003, HLA-A*3004, HLA-A*3201,HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204, HLA-A*0205, HLA-A*0206,HLA-A*0207, HLA-A*0214, HLA-A*0217, HLA-A*6802, HLA-A*6901, HLA-A*0301,HLA-A*1101, HLA-A*3101, HLA-A*3301, HLA-A*3303, HLA-A*6601, HLA-A*6801,HLA-A*7401, HLA-B*1501, HLA-B*1502, HLA-B*1512, HLA-B*1513, HLA-B*4601,HLA-B*5201).

In one embodiment the binding molecule is a MHC class II complex, ofHLA-type DP.

In one embodiment the binding molecule is a MHC class II complex, ofHLA-type DQ.

In one embodiment the binding molecule is a MHC class II complex, ofHLA-type DR.

In one embodiment the binding molecule is a MHC class II complex of HLAsupertype DR1 (eg. HLA-DR1*0101, HLA-DR*0102).

In one embodiment the binding molecule is a MHC class II complex of HLAsupertype DR3 (eg. HLA-DR1*1107, HLA-DR*0301, HLA-DR*0305, HLA-DR*0306,HLA-DR*0309).

In one embodiment the binding molecule is a MHC class II complex of HLAsupertype DR4 (eg. HLA-DR1*0401, HLA-DR*0402, HLA-DR*0404, HLA-DR*0405,HLA-DR*0408, HLA-DR*0410, HLA-DR*0426).

In one embodiment the binding molecule is a MHC class II complex of HLAsupertype DR7 (eg. HLA-DR1*0701).

In one embodiment the binding molecule is a MHC class II complex of HLAsupertype DR8 (eg. HLA-DR1*0801, HLA-DR*0802, HLA-DR*0804, HLA-DR*0806,HLA-DR*0813, HLA-DR*0817).

In one embodiment the binding molecule is a MHC class II complex of HLAsupertype DR11 (eg. HLA-DR1*1101, HLA-DR*1104, HLA-DR*1128, HLA-DR*1307,HLA-DR*1321).

In one embodiment the binding molecule is a MHC class II complex of HLAsupertype DR13 (eg. HLA-DR1*1102, HLA-DR*1114, HLA-DR*1120, HLA-DR*1301,HLA-DR*1304, HLA-DR*1322).

In one embodiment the binding molecule is a MHC class II complex of HLAsupertype DR15 (eg. HLA-DR1*1501, HLA-DR*1502).

In one embodiment the binding molecule is a MHC class II complex of HLAsupertype DR51 (eg. HLA-DR5*0101).

Generation of MHC Multimers

Different approaches to the generation of various types of MHC multimersare described in U.S. Pat. No. 5,635,363 (Altmann et al.), patentapplication WO 02/072631 A2 (Winther et al.), patent application WO99/42597, US patent 2004209295, U.S. Pat. No. 5,635,363. In brief, MHCmultimers can be generated by first expressing and purifying theindividual protein components of the MHC protein, and then combining theMHC protein components and the peptide, to form the MHC-peptide complex.Then an appropriate number of MHC-peptide complexes are linked togetherby covalent or non-covalent bonds to a multimerization domain. This canbe done by chemical reactions between reactive groups of themultimerization domain (e.g. vinyl sulfone functionalities on a dextranpolymer) and reactive groups on the MHC protein (e.g. amino groups onthe protein surface), or by non-covalent interaction between a part ofthe MHC protein (e.g. a biotinylated peptide component) and themultimerization domain (e.g. four binding sites for biotin on thestrepavidin tetrameric protein). As an alternative, the MHC multimer canbe formed by the non-covalent association of amino acid helices fused toone component of the MHC protein, to form a pentameric MHC multimer,held together by five helices in a coiled-coil structure making up themultimerization domain.

Appropriate chemical reactions for the covalent coupling of MHC and themultimerization domain include nucleophilic substitution by activationof electrophiles (e.g. acylation such as amide formation, pyrazoloneformation, isoxazolone formation; alkylation; vinylation; disulfideformation), addition to carbon-hetero multiple bonds (e.g. alkeneformation by reaction of phosphonates with aldehydes or ketones;arylation; alkylation of arenes/hetarenes by reaction with alkylboronates or enolethers), nucleophilic substitution using activation ofnucleophiles (e.g. condensations; alkylation of aliphatic halides ortosylates with enolethers or enamines), and cycloadditions.

Appropriate molecules, capable of providing non-covalent interactionsbetween the multimerization domain and the MHC-peptide complex, involvethe following molecule pairs and molecules: streptavidin/biotin,avidin/biotin, antibody/antigen, DNA/DNA, DNA/PNA, DNA/RNA, PNA/PNA,LNA/DNA, leucine zipper e.g. Fos/Jun, IgG dimeric protein, IgMmultivalent protein, acid/base coiled-coil helices, chelate/metalion-bound chelate, streptavidin (SA) and avidin and derivatives thereof,biotin, immunoglobulins, antibodies (monoclonal, polyclonal, andrecombinant), antibody fragments and derivatives thereof, leucine zipperdomain of AP-1 (jun and fos), hexa-his (metal chelate moiety), hexa-hatGST (glutathione S-transferase) glutathione affinity, Calmodulin-bindingpeptide (CBP), Strep-tag, Cellulose Binding Domain, Maltose BindingProtein, S-Peptide Tag, Chitin Binding Tag, Immuno-reactive Epitopes,Epitope Tags, E2Tag, HA Epitope Tag, Myc Epitope, FLAG Epitope, AU1 andAU5 Epitopes, Glu-Glu Epitope, KT3 Epitope, IRS Epitope, Btag Epitope,Protein Kinase-C Epitope, VSV Epitope, lectins that mediate binding to adiversity of compounds, including carbohydrates, lipids and proteins,e.g. Con A (Canavalia ensiformis) or WGA (wheat germ agglutinin) andtetranectin or Protein A or G (antibody affinity). Combinations of suchbinding entities are also comprised. In particular, when the MHC complexis tagged, the binding entity can be an “anti-tag”. By “anti-tag” ismeant an antibody binding to the tag and any other molecule capable ofbinding to such tag.

Generation of Components of MHC

When employing MHC multimers for diagnostic purposes, it is preferableto use a MHC allele that corresponds to the tissue type of the person oranimal to be diagnosed.

Once the MHC allele has been chosen, a peptide derived from theantigenic protein may be chosen. The choice will depend on factors suchas known or expected binding affinity of the MHC protein and the variouspossible peptide fragments that may be derived from the full sequence ofthe antigenic peptide, and will depend on the expected or known bindingaffinity and specificity of the MHC-peptide complex for the TCR.Preferably, the affinity of the peptide for the MHC molecule, and theaffinity and specificity of the MHC-peptide complex for the TCR, shouldbe high.

Similar considerations apply to the choice of MHC allele and peptide fortherapeutic and vaccine purposes. In addition, for some of theseapplications the effect of binding the MHC multimer to the TCR is alsoimportant. Thus, in these cases the effect on the T-cell's general statemust be considered, e.g. it must be decided whether the desired endresult is apoptosis or proliferation of the T-cell.

Likewise, it must be decided whether stability is important. For someapplications low stability may be an advantage, e.g. when a short-termeffect is desired; in other instances, a long-term effect is desired andMHC multimers of high stability is desired. Stabilities of the MHCprotein and of the MHC-peptide complex may be modified as describedelsewhere herein.

Finally, modifications to the protein structure may be advantageous forsome diagnostics purposes, because of e.g. increased stability, while infor vaccine purposes modifications to the MHC protein structure mayinduce undesired allergenic responses.

The generation of protein chains of MHC as well as the Stabilization ofMHC complexes is thoroughly explained in WO 2009/106073, which isincorporated herein by reference in its entirety.

Other TCR Binding Molecules

MHC I and MHC II complexes bind to TCRs. However, other molecules alsobind TCR. Some TCR-binding molecules are described in the following.

In one embodiment the binding molecule is an anti-target-moleculecapable of binding TCR.

In one embodiment the anti-target-molecule capable of binding TCR is amolecule that has homology to the classical MHC molecules and thereforepotentially could be TCR binding molecules. These other TCR binding orMHC like molecules include:

Non-Classical MHC Complexes and other MHC-Like Molecules

Non-classical MHC complexes include protein products of MHC Ib and MHCIIb genes. MHC Ib genes encode β2m-associated cell-surface molecules butshow little polymorphism in contrast to classical MHC class I genes.Protein products of MHC class Ib genes include HLA-E, HLA-G, HLA-F,HLA-H, MIC A, MIC B, ULBP-1, ULBP-2, ULBP-3 in humans and H2-M, H2-Q,H2-T and Rae1 in mice.

Non-classical MHC II molecules (protein products of MHC IIb genes)include HLA-DM, HLA-DO in humans and H2-DM and H2-DO in mice that areinvolved in regulation of peptide loading into MHC II molecules.

Another MHC-like molecule of special interest is the MHC I-like moleculeCD1. CD1 is similar to MHC I molecules in its organization of subunitsand association with β2m but presents glycolipids and lipids instead ofpeptides.

Artificial Molecules Capable of Binding Specific TCRs

Of special interest are antibodies that bind TCRs. Antibodies hereininclude full length antibodies of isotype IgG, IgM, IgE, IgA andtruncated versions of these, antibody fragments like Fab fragments andscFv. Antibodies also include antibodies of antibody fragments displayedon various supramolecular structures or solid supports, includingfilamentous phages, yeast, mammalian cells, fungi, artificial cells ormicelles, and beads with various surface chemistries.

Peptide Binding TCR

Another embodiment of special interest is peptides that bind TCRs.Peptides herein include peptides composed of natural, non-natural and/orchemically modified amino acids with a length of 8-20 amino acid. Thepeptides could also be longer than 20 amino acids or shorter than 8amino acids. The peptides can or cannot have a defined tertiarystructure.

Aptamers

Aptamers are another preferred group of TCR ligands. Aptamers are hereinunderstood as natural nucleic acids (e.g. RNA and DNA) or unnaturalnucleic acids (e.g. PNA, LNA, morpholinos) capable of binding TCR. Theaptamer molecules consist of natural or modified nucleotides in variouslengths.

Other TCR-binding molecules can be ankyrin repeat proteins or otherrepeat proteins, Avimers, or small chemical molecules, as long as theyare capable of binding TCR with a dissociation constant smaller than10⁻³ M.

BM: Anti-Target Molecules

In one embodiment the binding molecule is an anti-target-moleculecapable of associating with, recognizing and/or binding to apredetermined target structure belonging to or associated with anentity/cell, e.g. in a sample. A typical target for a binding moleculeis a ‘marker molecule’, which marker molecule is specific for a givencell or cell type.

Any binding molecule that is capable of specifically associating with,recognizing and/or binding to a predetermined target structure isencompassed within the present invention.

In one embodiment the binding molecule is an anti-target-moleculecapable of binding a specific cell type.

In one embodiment the binding molecule is an anti-target-moleculecapable of binding a specific cell type selected from the groupconsisting of immune cells, lymphocytes, monocytes, dendritic cells,T-cells, B-cells and NK cells.

In one embodiment the binding molecule is an anti-target-moleculecapable of binding a specific cell type selected from the groupconsisting CD4+ T cells, CD8+ T cells, αβ T cells, invariant γδ T cellsand antigen-specific T-cells.

In one embodiment the binding molecule is an anti-target-moleculecapable of binding a cell comprising TCRs. In one embodiment the bindingmolecule is an anti-target-molecule capable of binding a cell comprisingBCRs.

In one embodiment the binding molecule is an anti-target-moleculecapable of binding a specific cancer cell.

In one embodiment the binding molecule is an anti-target-moleculecapable of binding a target specifically associated with an organselected from the group consisting of lymph nodes, kidney, liver, skin,brain, heart, muscles, bone marrow, skin, skeleton, lungs, therespiratory tract, spleen, thymus, pancreas, exocrine glands, bladder,endocrine glands, reproduction organs including the phallopian tubes,eye, ear, vascular system, the gastroinstestinal tract including smallintestines, colon, rectum, canalis analis and prostate gland.

Anti-target-molecule is in one embodiment selected from antibodies,antibody mimetics, natural ligands, variants or fragments of naturalligands, synthetic ligands, agonists, antagonists, aptamers (includingDNA aptamers, RNA aptamers and peptide aptamers), peptides, andartificial molecules capable of binding a specific target.

Peptide Aptamer

In one embodiment the binding molecule is an anti-target-molecule. Inone embodiment the anti-target-molecule is a peptide aptamer, such as apeptide that bind a target. Peptides herein include peptides composed ofnatural, non-natural and/or chemically modified amino acids, in oneembodiment with a length of 8-20 amino acid, such as 5-25 amino acids,for example 5-40 amino acids. The peptides can or cannot have a definedtertiary structure.

In one embodiment the binding molecule is a peptide of 1-100 amino acidresidues without tertiary structure.

Nucleic Acid Aptamer

In one embodiment the binding molecule is an anti-target-molecule. Inone embodiment the anti-target-molecule is a nucleic acid aptamer (oroligonucleotide aptamer, or simply ‘oligonucleotide’). Nucleic acidaptamers are herein understood as natural nucleic acids (e.g. RNA andDNA) or artificial nucleic acids (e.g. PNA, LNA, morpholinos) capable ofbinding a target. The aptamer molecules consist of natural or modifiednucleotides in various lengths.

Other target-binding molecules include ankyrin repeat proteins or otherrepeat proteins, Avimers, or small chemical molecules, as long as theyare capable of binding the target with an acceptable dissociationconstant such as smaller than 10⁻³ M.

Ligand

In one embodiment the binding molecule is an anti-target-molecule. Inone embodiment the anti-target-molecule is a ligand capable of binding atarget.

Antibodies

In one embodiment the binding molecule is an anti-target-molecule. Inone embodiment anti-target-molecule is an antibody. In a preferredembodiment the anti-target-molecule is an antibody that selectivelyassociates with, recognizes and/or binds to a predetermined targetstructure belonging to or associated with an entity/cell in a sample.

Immunoglobulins are a class of structurally related glycoproteinsconsisting of two pairs of polypeptide chains, one pair of light (L) lowmolecular weight chains and one pair of heavy (H) chains, all fourinter-connected by disulfide bonds. The structure of immunoglobulins hasbeen well characterized. Briefly, each heavy chain typically iscomprised of a heavy chain variable region (VH) and a heavy chainconstant region (CH) typically comprised of three domains, CH1, CH2, andCH3. Each light chain typically is comprised of a light chain variableregion (VL) and a light chain constant region, typically comprised ofone domain (CL). The VH and VL regions may be further subdivided intoregions of hypervariability also termed complementarity determiningregions (CDRs), interspersed with regions that are more conserved,termed framework regions (FRs).

The term “antibody” or “Ab” in the context of the present inventionrefers to an immunoglobulin molecule, a fragment of an immunoglobulinmolecule, or a derivative of either thereof, which has the ability tospecifically bind to an antigen under typical physiological conditions.The variable regions of the heavy and light chains of the immunoglobulinmolecule contain a binding domain that interacts with an antigen. Anantibody may also be multispecific, having specificities for two or moredifferent epitopes, typically non-overlapping. Examples of multispecificantibodies include bispecific antibodies, diabodies, and similarantibody molecules. As indicated above, the term antibody herein, unlessotherwise stated, includes fragments of an antibody that retain theability to specifically bind to the antigen. The antigen-bindingfunction of an antibody may be performed by fragments of a full-lengthantibody, e.g., Fab and F(ab′)2 fragments. In the context of the presentinvention the term antibody, unless specified otherwise, includespolyclonal antibodies, monoclonal antibodies (mAbs), antibody-likepolypeptides such as chimeric antibodies and humanized antibodies. Anantibody as generated can possess any isotype. The term “epitope” meansa protein determinant capable of specific binding to an antibody.

The terms “human antibody” include antibodies having variable andconstant regions derived from human germline immunoglobulin sequences.The human antibodies of the invention may include amino acid residuesnot encoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo). However, the term “human antibody”, as used herein,is not intended to include antibodies in which CDR sequences derivedfrom the germline of another mammalian species, such as a mouse, havebeen grafted onto human framework sequences.

The terms “monoclonal antibody” (mAb), refer to a preparation ofantibody molecules of single molecular composition. A monoclonalantibody composition displays a single binding specificity and affinityfor a particular epitope. Accordingly, the term “human monoclonalantibody” refers to antibodies displaying a single binding specificitywhich have variable and constant regions derived from human germlineimmunoglobulin sequences. The human monoclonal antibodies may beproduced by a hybridoma which includes a B cell obtained from atransgenic or transchromosomal non-human animal, such as a transgenicmouse, having a genome comprising a human heavy chain transgene and alight chain transgene, fused to an immortalized cell.

As used herein, “isotype” refers to the immunoglobulin class (forinstance IgGI, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) that is encodedby heavy chain constant region genes. The term “full-length antibody”when used herein, refers to an antibody which contains all heavy andlight chain constant and variable domains that are normally found in anantibody of that isotype.

Antibody Mimetics

In one embodiment the binding molecule is an anti-target-molecule. Inone embodiment anti-target-molecule is an antibody mimetic. Antibodymimetics are organic compounds that, like antibodies, can specificallybind antigens, but that are not structurally related to antibodies.

Antibody mimetics include, but is not limited to, affibody molecules,affilinns, affimers, affitins, alphabodies, anticalins, avimers,DARPins, fynomers, Kunitz domain peptides and monobodies.

Avimers (short for avidity multimers) are artificial proteins that areable to specifically bind to certain antigens via multiple bindingsites. Avimers consist of two or more peptide sequences of 30 to 35amino acids each, connected by linker peptides.

Targets of Binding Molecules

In one embodiment the binding molecule is an anti-target-moleculecapable of associating with, recognizing and/or binding to apredetermined target structure belonging to or associated with anentity/cell in a sample.

In one embodiment the target for a binding molecule is a ‘markermolecule’, which marker molecule is specific for a given cell or celltype.

In one embodiment the target is an intracellular target.

In one embodiment the target is a cell-surface target.

In one embodiment the target is a membrane-associated target.

In one embodiment the target is a receptor.

In one embodiment the receptor is an intracellular receptor.

In one embodiment the receptor is a cell-surface receptor ormembrane-associated receptor.

In one embodiment the target is a soluble receptor.

In one embodiment the target is a extracellular receptor.

In one embodiment the binding molecule is an anti-target-moleculecapable of binding a target selected from the group consisting of CD1,CD1a, CD1b, CD1c, CD1d, and MR1.

In one embodiment the target is a T cell receptor.

In one embodiment the target is a B cell receptor.

In one embodiment the target is CD4.

In one embodiment the target is CD8.

In one embodiment the target is CD20.

In one embodiment the target of the binding molecule (anti-targetmolecule) binds a target selected from the group consisting of cancercell markers, developmental markers, stem cell markers, cell cyclemarkers, proliferation markers, activation markers, hormones, hormonereceptors, cluster of differentiation (CD), cytokines and cytokinereceptors.

In one embodiment the cluster of differentiation is selected from agroup consisting of CD1-10, CD10-20, CD20-30, CD30-40, CD40-50,CD50-100, CD100-200, CD200-300 and CD300-364.

In one embodiment the cancer cell marker is selected from a groupconsisting of HER2, CA125, Tyrosinase, Melanoma-associated antigen(MAGE), abnormal products of Ras or p53, Carcinoembryonic antigen,Muc-1, Epithelial tumor antigen, Carbonic Anhydrase, VEGFR, EGFR, TRAIand RANKL.

In one embodiment the stem cell marker is selected from a groupconsisting of Stro-1, CD146, CD105, CD44, c-kit, Oct4, Sox-2, Klf4,EphB, Nestin and TWIST-1.

In one embodiment the developmental cell marker is selected from a groupconsisting of Nanog, Oct4, Sox2, TEKT-1, NANOS, c-kit, Sox9, Notch,Msx1, Msx2 and Col1.

In one embodiment the cytokine is selected from a group consisting ofTNFα, TNFβ, TNF, IFNα, IFNβ, IFNγ, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-10-20, IL-20-30, IL-30, IL-31, IL-32, IL-33,IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, NFκB, chemokinesincluding CC chemokines (CCL1-CCL-28), CXC chemokines (CXCL1-CXCL17) Cchemokines (XCL-1 and -2) and CX3X chemokines (CX3CL1).

In one embodiment the proliferation marker is selected from a groupconsisting of CyclinA, CyclinB, PCNA, PC10, p53, Mdm2, Cyclin D, CyclinE, Rb, ARF and HDM2.

In one embodiment the activation marker is selected from a groupconsisting of CD28, Tbet, Eomes, Blimp, Bcl-6, CD27, MHC-II, TNF, IFN,Fizz1, ARG1 and CCL22R.

In one embodiment the hormone is selected from a group consisting ofestrogen, PTH, ADH, T3, ANP, Epinephrine, Norepinephrine, Cortisol,Corticosterone, Aldosterone, Progestin, EPO, Leptin, Insulin, Glucagon,T4, ACTH, FSH, oxytocin and Calcitriol.

In one embodiment the hormone receptor is selected from a groupconsisting of EstrogenR (ER), GLP-1R, Thyroid receptor, Leptin receptor,Epinephrine receptor, Insulin receptor and Glucagon receptor.

In one embodiment the intracellular marker is selected from a groupconsisting of Cyclins, Cytokines and organelle markers (for exampleApg12, Syntaxin, PAF-46, Histones, Early endosome antigen, clathrin,tubulins, PAF49, FTCD).

In one embodiment the target of the binding molecule is selected fromthe group consisting of CD2, CD3, CD4, CD5, CD8, CD9, CD27, CD28, CD30,CD69, CD134 (OX40), CD137 (4-1BB), CD147, CDw150 (SLAM), CD152 (CTLA-4),CD153 (CD30L), CD40L (CD154), NKG2D, ICOS, HVEM, HLA Class II, PD-1, Fas(CD95), FasL, CD40, CD48, CD58, CD70, CD72, B7.1 (CD80), B7.2 (CD86),B7RP-1, B7-H3, PD-L1, PD-L2, CD134L, CD137L, ICOSL, LIGHT CD16, NKp30,NKp44, NKp46, NKp80, 2B4, KIR, LIR, CD94/NKG2A, CD94/NKG2C, LFA-1,CD11a/18, CD54 (ICAM-1), CD106 (VCAM), CD49a,b,c,d,e,f/CD29 (VLA-4),CD11a, CD14, CD15, CD19, CD25, CD30, CD37, CD49a, CD49e, CD56, CD27,CD28, CD45, CD45RA, CD45RO, CD45RB, CCR7, CCRS, CD62L, CD75, CD94, CD99,CD107b, CD109, CD152, CD153, CD154, CD160, CD161, CD178, CDw197, CDw217,Cd229, CD245, CD247 and Foxp3.

Additional Components of Detection Molecule

Additional components or substituents may be coupled to the detectionmolecule, either coupled to carrier or added as individual componentsnot coupled to carrier.

In one embodiment the detection molecule further comprises an enzymecapable of catalysing the transfer of a cell surface moiety from a cellsurface entity to the binding molecule of the detection molecule,wherein said cell surface moiety binds to (or associates with) thebinding molecule.

In one embodiment the cell surface entity is a cell surface protein. Inone embodiment the cell surface moiety is a peptide fragment or ‘peptidetag’.

The peptide tag added to the binding molecule can be used as a means ofisolating the detection molecules that have been in contact with saidcell surface entity.

Attachment of Biologically Active Molecules to Binding Molecules

Engagement of MHC complex to the specific T cell receptor leads to asignaling cascade in the T cell. However, T-cells normally respond to asingle signal stimulus by going into apoptosis. T cells needs a secondsignal in order to become activated and start development into aspecific activation state e.g. become an active cytotoxic T cell, helperT cell or regulatory T cell.

It is to be understood that the binding molecule such as MHC complex orMHC multimer of the invention may further comprise one or moreadditional substituents, such as biologically active molecules. Thedefinition of the terms “one or more”, “a plurality”, “a”, “an”, and“the” also apply here. Such biologically active molecules may beattached to the construct in order to affect the characteristics of theconstructs, e.g. with respect to binding properties, effects, MHCmolecule specificities, solubility, stability, or detectability. Forinstance, spacing could be provided between the two or more bindingmolecules such as two or more MHC complexes, one or both chromophores ofa Fluorescence Resonance Energy Transfer (FRET) donor/acceptor paircould be inserted, functional groups could be attached, or groups havinga biological activity could be attached.

Binding molecules such as MHC multimers can be covalently ornon-covalently associated with various molecules: having adjuvanteffects; being immune targets e.g. antigens; having biological activitye.g. enzymes, regulators of receptor activity, receptor ligands, immunepotentiators, drugs, toxins, co-receptors, proteins and peptides ingeneral; sugar moieties; lipid groups; nucleic acids including siRNA;nano particles; small molecules. In the following these molecules arecollectively called biologically active molecules. Such molecules can beattached to the binding molecule such as MHC multimer using the sameprinciples as those described for attachment of binding molecule such asMHC complexes to multimerisation domains as described elsewhere herein.In brief, attachment can be done by chemical reactions between reactivegroups on the biologically active molecule and reactive groups of themultimerisation domain and/or between reactive groups on thebiologically active molecule and reactive groups of the binding moleculesuch as MHC-peptide complex. Alternatively, attachment is done bynon-covalent interaction between part of the multimerisation domain andpart of the biological active molecule or between part of the bindingmolecule such as MHC-peptide complex and part of the biological activemolecule. In both covalent and non-covalent attachment of thebiologically molecule to the multimerisation domain a connector moleculecan connect the two. The connector molecule can be covalent ornon-covalent attached to both molecules. Examples of connector moleculesare described elsewhere herein. Some of the binding molecules such asMHCmer structures better allow these kinds of modifications than others.Biological active molecules can be attached repetitively aiding torecognition by and stimulation of the innate immune system via Toll orother receptors. Binding molecules such as MHC multimers carrying one ormore additional groups can be used as therapeutic or vaccine reagents.

In particular, the biologically active molecule may be selected from:

-   -   proteins such as MHC Class I-like proteins like MIC A, MIC B,        CD1d, HLA E, HLA F, HLA G, HLA H, ULBP-1, ULBP-2, and ULBP-3,    -   co-stimulatory molecules such as CD2, CD3, CD4, CD5, CD8, CD9,        CD27, CD28, CD30, CD69, CD134 (OX40), CD137 (4-1BB), CD147,        CDw150 (SLAM), CD152 (CTLA-4), CD153 (CD30L), CD40L (CD154),        NKG2D, ICOS, HVEM, HLA Class II, PD-1, Fas (CD95), FasL        expressed on T and/or NK cells, CD40, CD48, CD58, CD70, CD72,        B7.1 (CD80), B7.2 (CD86), B7RP-1, B7-H3, PD-L1, PD-L2, CD134L,        CD137L, ICOSL, LIGHT expressed on APC and/or tumour cells,    -   cell modulating molecules such as CD16, NKp30, NKp44, NKp46,        NKp80, 2B4, KIR, LIR, CD94/NKG2A, CD94/NKG2C expressed on NK        cells, IFN-alpha, IFN-beta, IFN-gamma, IL-1, IL-2, IL-3, IL-4,        IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-15, CSFs        (colony-stimulating factors), vitamin D3, IL-2 toxins,        cyclosporin, FK-506, rapamycin, TGF-beta, clotrimazole,        nitrendipine, and charybdotoxin,    -   accessory molecules such as LFA-1, CD11a/18, CD54 (ICAM-1),        CD106 (VCAM), and CD49a,b,c,d,e,f/CD29 (VLA-4),    -   adhesion molecules such as ICAM-1, ICAM-2, GlyCAM-1, CD34,        anti-LFA-1, anti-CD44, anti-beta7, chemokines, CXCR4, CCRS,        anti-selectin L, anti-selectin E, and anti-selectin P,    -   toxic molecules selected from toxins, enzymes, antibodies,        radioisotopes, chemiluminescent substances, bioluminescent        substances, polymers, metal particles, and haptens, such as        cyclophosphamide, methrotrexate, Azathioprine, mizoribine,        15-deoxuspergualin, neomycin, staurosporine, genestein,        herbimycin A, Pseudomonas exotoxin A, saporin, Rituxan, Ricin,        gemtuzumab ozogamicin, Shiga toxin, heavy metals like inorganic        and organic mercurials, and FN18-CRM9, radioisotopes such as        incorporated isotopes of iodide, cobalt, selenium, tritium, and        phosphor, and haptens such as DNP, and digoxiginin,

and combinations of any of the foregoing, as well as antibodies(monoclonal, polyclonal, and recombinant) to the foregoing, whererelevant. Antibody derivatives or fragments thereof may also be used.

Biological active molecules as described above may also be attached toantigenic peptide products or antigenic polypeptide products using sameprinciples for attachment.

Linker

The linker comprised in the detection molecule of the present inventionis a molecular entity and/or bond that connect the binding molecule andthe label of the detection molecule. The connection may be direct orindirect, and may comprise a covalent or non-covalent binding. A linkeraccording to the present invention comprises, or is identical to, acarrier molecule (or carrier), a multimerization domain and/or abackbone, and may equally be referred to as such herein.

In a preferred embodiment the linker consist of or comprises one or moremolecules chosen from the group of dextran, streptavidin, peptide, orantibody.

In a preferred embodiment the linker connecting the binding molecule andthe label has a length of 1-2 Å, 3-4 Å, 5-8 Å, 9-15 Å, 16-30 Å, 31-50 Å,51-100 Å, 101-200 Å, 201-500 Å, 501-2000 Å, or longer than 2000 Å.

In one embodiment the linker of the invention, connecting the bindingmolecule and the label, consists or comprises one or more of thefollowing entities: a short peptide, a modified or non-modified alkane,alkene or alkyne, a polyamide, ethylenglycol or polyethylenglycol, asmall chemical entity such as an amide bond or other chemical bond, anoligonucleotide, biotin, the bond formed after reaction of a thiol withan NHS-ester or a maleimide.

Multimerisation Domain

In a preferred aspect of the invention one or multiple binding molecules(BM), as well as one or more labels, associate with a multimerizationdomain to form a detection molecule,

Wherein at least two binding molecules are associated via amultimerization domain this constitutes a binding molecule multimer (BMmultimer).

The term ‘multimerization domain’ is used to refer to a type of linkerin the below; namely any molecular entity and/or bond that connect thebinding molecule and the label of the detection molecule; alone or via aconnector.

A number of binding molecules associate with one or more multimerizationdomains to form a detection molecule comprising one or more bindingmolecules and one or more labels. In one embodiment a number of MHCcomplexes (binding molecules) associate with a multimerization domain toform a MHC multimer.

The size of the multimerization domain spans a wide range, frommultimerisation domains based on small organic molecule scaffolds tolarge multimers based on a cellular structure or solid support. Themultimerization domain may thus be based on different types of carriersor scaffolds, and likewise, the attachment of binding molecules, such asMHC complexes, to the multimerization domain may involve covalent ornon-covalent connectorss. Characteristics of different kinds ofmultimerization domains are described below.

Molecular Weight of Multimerization Domain.

-   -   In one embodiment the multimerization domain(s) in the present        invention is preferably less than 1,000 Da (small molecule        scaffold). Examples include short peptides (e.g. comprising 10        amino acids), and various small molecule scaffolds (e.g.        aromatic ring structures).    -   In another embodiment the multimerization domain(s) is        preferably between 1,000 Da and 10,000 Da (small molecule        scaffold, small peptides, small polymers). Examples include        polycyclic structures of both aliphatic and aromatic compounds,        peptides comprising e.g. 10-100 amino acids, and other polymers        such as dextran, polyethylenglycol, and polyureas.    -   In another embodiment the multimerization domain(s) is between        10,000 Da and 100,000 Da (Small molecule scaffold, polymers e.g.        dextran, streptavidin, IgG, pentamer structure). Examples        include proteins and large polypeptides, small molecule        scaffolds such as steroids, dextran, dimeric streptavidin, and        multi-subunit proteins such as used in Pentamers.    -   In another embodiment the multimerization domain(s) is        preferably between 100,000 Da and 1,000,000 Da (Small molecule        scaffold, polymers e.g. dextran, streptavidin, IgG, pentamer        structure). Typical examples include larger polymers such as        dextran (used in e.g. Dextramers), and streptavidin tetramers.    -   In another embodiment the multimerization domain(s) is        preferably larger than 1,000,000 Da (Small molecule scaffold,        polymers e.g. dextran, streptavidin, IgG, pentamer structure,        cells, liposomes, artificial lipid bilayers, polystyrene beads        and other beads. Most examples of this size involve cells or        cell-based structures such as micelles and liposomes, as well as        beads and other solid supports.

As mentioned elsewhere herein multimerisation domains can comprisecarrier molecules (connectors), scaffolds or combinations of the two.

Type of Multimerization Domain.

In principle any kind of carrier or scaffold can be used asmultimerization domain, including any kind of cell, polymer, protein orother molecular structure, or particles and solid supports. Belowdifferent types and specific examples of multimerization domains arelisted.

-   -   Cell. Cells can be used as carriers. Cells can be either alive        and mitotic active, alive and mitotic inactive as a result of        irradiation or chemically treatment, or the cells may be dead.        The MHC expression may be natural (i.e. not stimulated) or may        be induced/stimulated by e.g. Inf-γ. Of special interest are        natural antigen presenting cells (APCs) such as dendritic cells,        macrophages, Kupfer cells, Langerhans cells, B-cells and any MHC        expressing cell either naturally expressing, being transfected        or being a hybridoma.    -   Cell-like structures. Cell-like carriers include membrane-based        structures carrying MHC-peptide complexes in their membranes        such as micelles, liposomes, and other structures of membranes,        and phages such as filamentous phages.    -   Solid support. Solid support includes beads, particulate matters        and other surfaces. A preferred embodiment include beads        (magnetic or non-magnetic beads) that carry electrophilic groups        e.g. divinyl sulfone activated polysaccharide, polystyrene beads        that have been functionalized with tosyl-activated esters,        magnetic polystyrene beads functionalized with tosyl-activated        esters), and where binding molecules such as MHC complexes may        be covalently immobilized to these by reaction of nucleophiles        comprised within the BM such as the MHC complex with the        electrophiles of the beads. Beads may be made of sepharose,        sephacryl, polystyrene, agarose, polysaccharide, polycarbamate        or any other kind of beads that can be suspended in aqueous        buffer.    -   Another embodiment includes surfaces, i.e. solid supports and        particles carrying immobilized binding molecules such as MHC        complexes on the surface. Of special interest are wells of a        microtiter plate or other plate formats, reagent tubes, glass        slides or other supports for use in microarray analysis, tubings        or channels of micro fluidic chambers or devices, Biacore chips        and beads    -   Molecule. Multimerization domains may also be molecules or        complexes of molecules held together by non-covalent bonds. The        molecules constituting the multimerization domain can be small        organic molecules or large polymers, and may be flexible linear        molecules or rigid, globular structures such as e.g. proteins.        Different kinds of molecules used in multimerization domains are        described below.        -   Small organic molecules. Small organic molecules here            includes steroids, peptides, linear or cyclic structures,            and aromatic or aliphatic structures, and many others. The            prototypical small organic scaffold is a functionalized            benzene ring, i.e. a benzene ring functionalized with a            number of reactive groups such as amines, to which a number            of binding molecules such as MHC molecules may be covalently            linked. However, the types of reactive groups constituting            the connector connecting the binding molecules such as MHC            complex and the multimerization domain, as well as the type            of scaffold structure, can be chosen from a long list of            chemical structures. A non-comprehensive list of scaffold            structures are listed below.        -   Typical scaffolds include aromatic structures,            benzodiazepines, hydantoins, piperazines, indoles, furans,            thiazoles, steroids, diketopiperazines, morpholines,            tropanes, coumarines, qinolines, pyrroles, oxazoles, amino            acid precursors, cyclic or aromatic ring structures, and            many others.        -   Typical carriers include linear and branched polymers such            as peptides, polysaccharides, nucleic acids, and many            others. Multimerization domains based on small organic or            polymer molecules thus include a wealth of different            structures, including small compact molecules, linear            structures, polymers, polypeptides, polyureas,            polycarbamates, cyclic structures, natural compound            derivatives, alpha-, beta-, gamma-, and omega-peptides,            mono-, di- and tri-substituted peptides, L- and D-form            peptides, cyclohexane- and cyclopentane-backbone modified            beta-peptides, vinylogous polypeptides, glycopolypeptides,            polyamides, vinylogous sulfonamide peptide,            Polysulfonamide-conjugated peptide (i.e., having prosthetic            groups), Polyesters, Polysaccharides such as dextran and            aminodextran, polycarbamates, polycarbonates, polyureas,            poly-peptidylphosphonates, Azatides, peptoids (oligo            N-substituted glycines), Polyethers, ethoxyformacetal            oligomers, poly-thioethers, polyethylene, glycols (PEG),            polyethylenes, polydisulfides, polyarylene sulfides,            Polynucleotides, PNAs, LNAs, Morpholinos, oligo pyrrolinone,            polyoximes, Polyimines, Polyethyleneimine, Polyacetates,            Polystyrenes, Polyacetylene, Polyvinyl, Lipids,            Phospholipids, Glycolipids, polycycles, (aliphatic),            polycycles (aromatic), polyheterocycles, Proteoglycan,            Polysiloxanes, Polyisocyanides, Polyisocyanates,            polymethacrylates, Monofunctional, Difunctional,            Trifunctional and Oligofunctional open-chain hydrocarbons,            Monofunctional, Difunctional, Trifunctional and            Oligofunctional Nonaromat Carbocycles, Monocyclic, Bicyclic,            Tricyclic and Polycyclic Hydrocarbons, Bridged Polycyclic            Hydrocarbones, Monofunctional, Difunctional, Trifunctional            and Oligofunctional Nonaromatic, Heterocycles, Monocyclic,            Bicyclic, Tricyclic and Polycyclic Heterocycles, bridged            Polycyclic Heterocycles, Monofunctional, Difunctional,            Trifunctional and Oligofunctional Aromatic Carbocycles,            Monocyclic, Bicyclic, Tricyclic and Polycyclic Aromatic            Carbocycles, Monofunctional, Difunctional, Trifunctional and            Oligofunctional Aromatic Hetero-cycles. Monocyclic,            Bicyclic, Tricyclic and Polycyclic Heterocycles. Chelates,            fullerenes, and any combination of the above and many            others.        -   Biological polymers. Biological molecules here include            peptides, proteins (including antibodies, coiled-coil            helices, streptavidin and many others), nucleic acids such            as DNA and RNA, and polysaccharides such as dextran. The            biological polymers may be reacted with MHC complexes (e.g.            a number of MHC complexes chemically coupled to e.g. the            amino groups of a protein), or may be linked through e.g.            DNA duplex formation between a carrier DNA molecule and a            number of DNA oligonucleotides each coupled to a MHC            complex. Another type of multimerization domain based on a            biological polymer is the streptavidin-based tetramer, where            a streptavidin binds up to four biotinylated MHC complexes,            as described above (see Background of the invention).        -   Self-assembling multimeric structures. Several examples of            commercial MHC multimers exist where the multimer is formed            through self-assembling. Thus, the Pentamers are formed            through formation of a coiled-coil structure that holds            together 5 MHC complexes in an apparently planar structure.            In a similar way, the Streptamers are based on the            Streptactin protein which oligomerizes to form a MHC            multimer comprising several MHC complexes.

In the following, alternative ways to make binding molecules such as MHCmultimers based on a molecule multimerization domain are described. Theyinvolve one or more of the above-mentioned types of multimerizationdomains.

MHC dextramers can be made by coupling MHC complexes to dextran via astreptavidin-biotin interaction. In principle, biotin-streptavdin can bereplaced by any dimerization domain, where one half of the dimerizationdomain is coupled to the MHC complex and the other half is coupled todextran. For example, an acidic helix (one half of a coiled-coil dimer)is coupled or fused to MHC, and a basic helix (other half of acoiled-coil dimmer) is coupled to dextran. Mixing the two results in MHCbinding to dextran by forming the acid/base coiled-coil structure.

Binding molecule dextramers can be made by coupling binding molecules todextran via a streptavidin-biotin interaction. In principle,biotin-streptavdin can be replaced by any dimerization domain, where onehalf of the dimerization domain is coupled to the binding molecule andthe other half is coupled to dextran. For example, an acidic helix (onehalf of a coiled-coil dimer) is coupled or fused to the BM, and a basichelix (other half of a coiled-coil dimmer) is coupled to dextran. Mixingthe two results in the BM binding to dextran by forming the acid/basecoiled-coil structure.

Antibodies can be used as scaffolds by using their capacity to bind to acarefully selected antigen found naturally or added as a tag to a partof the binding molecule such as the MHC molecule not involved in peptidebinding. For example, IgG and IgE will be able to bind two bindingmolecules such as two MHC molecules, and IgM having a pentamericstructure will be able to bind 10 binding molecules such as 10 MHCmolecules. The antibodies can be full-length or truncated; a standardantibody-fragment includes the Fab2 fragment.

Peptides involved in coiled-coil structures can act as scaffold bymaking stable dimeric, trimeric, tetrameric and pentameric interactions.Examples hereof are the Fos-Jun heterodimeric coiled coil, the E. colihomo-trimeric coiled-coil domain Lpp-56, the engineered Trp-zipperprotein forming a discrete, stable, α-helical pentamer in water atphysiological pH.

Further examples of suitable scaffolds, carriers and connectors arestreptavidin (SA) and avidin and derivatives thereof, biotin,immunoglobulins, antibodies (monoclonal, polyclonal, and recombinant),antibody fragments and derivatives thereof, leucine zipper domain ofAP-1 (jun and fos), hexa-his (metal chelate moiety), hexa-hat GST(glutathione S-tranferase), glutathione, Calmodulin-binding peptide(CBP), Strep-tag, Cellulose Binding Domain, Maltose Binding Protein,S-Peptide Tag, Chitin Binding Tag, Immuno-reactive Epitopes, EpitopeTags, E2Tag, HA Epitope Tag, Myc Epitope, FLAG Epitope, AU1 and AU5Epitopes, Glu-Glu Epitope, KT3 Epitope, IRS Epitope, Btag Epitope,Protein Kinase-C Epitope, VSV Epitope, lectins that mediate binding to adiversity of compounds, including carbohydrates, lipids and proteins,e.g. Con A (Canavalia ensiformis) or WGA (wheat germ agglutinin) andtetranectin or Protein A or G (antibody affinity). Combinations of suchbinding entities are also comprised. Non-limiting examples arestreptavidin-biotin and jun-fos. In particular, when the MHC molecule istagged, the binding entity may be an “anti-tag”. By “anti-tag” is meantan antibody binding to the tag, or any other molecule capable of bindingto such tag.

Wherein the binding molecule comprise MHC complexes, these can bemultimerized by other means than coupling or binding to amultimerization domain. Thus, the multimerization domain may be formedduring the multimerization of MHCs. One such method is to extend thebound antigenic peptide with dimerization domains. One end of theantigenic peptide is extended with dimerization domain A (e.g. acidichelix, half of a coiled-coil dimer) and the other end is extended withdimerization domain B (e.g. basic helix, other half of a coiled-coildimer). When MHC complexes are loaded/mixed with these extended peptidesthe following multimer structure will be formed: A-MHC-BA-MHC-BA-MHC-Betc. The antigenic peptides in the mixture can either be identical or amixture of peptides with comparable extended dimerization domains.Alternatively both ends of a peptide are extended with the samedimerization domain A and another peptide (same amino acid sequence or adifferent amino acid sequence) is extended with dimerization domain B.When MHC and peptides are mixed the following structures are formed:A-MHC-AB-MHC-BA-MHC-AB-MHC-B etc. Multimerization of MHC complexes byextension of peptides are restricted to MHC II molecules since thepeptide binding groove of MHC I molecules is typically closed in bothends thereby limiting the size of peptide that can be embedded in thegroove, and therefore preventing the peptide from extending out of thegroove.

Another multimerization approach applicable to both MHC I and MHC IIcomplexes is based on extension of N- and C-terminal of the MHC complex.For example the N-terminal of the MHC complex is extended withdimerization domain A and the C-terminal is extended with dimerizationdomain B. When MHC complexes are incubated together they pair with eachother and form multimers like: A-MHC-BA-MHC-BA-MHC-BA-MHC-B etc.Alternatively the N-terminal and the C-terminal of a MHC complex areboth extended with dimerization domain A and the N-terminal andC-terminal of another preparation of MHC complex (either the same or adifferent MHC) are extended with dimerization domain B. When these twotypes of MHC complexes are incubated together multimers will be formed:A-MHC-AB-MHC-BA-MHC-AB-MHC-B etc.

In all the above-described examples the extension can be eitherchemically coupled to the peptide/MHC complex or introduced as extensionby gene fusion. Dimerization domain AB can be any molecule pair able tobind to each other, such as acid/base coiled-coil helices,antibody-antigen, DNA-DNA, PNA-PNA, DNA-PNA, DNA-RNA, LNA-DNA, leucinezipper e.g. Fos/Jun, streptavidin-biotin and other molecule pairs asdescribed elsewhere herein.

In one embodiment the one or more multimerization domains each have amolecular weight of from 50,000 Da to preferably less than 1,000,000 Da,such as from 50,000 Da to 980,000; for example from 50,000 Da to960,000; such as from 50,000 Da to 940,000; for example from 50,000 Dato 920,000; such as from 50,000 Da to 900,000; for example from 50,000Da to 880,000; such as from 50,000 Da to 860,000; for example from50,000 Da to 840,000; such as from 50,000 Da to 820,000; for examplefrom 50,000 Da to 800,000; such as from 50,000 Da to 780,000; forexample from 50,000 Da to 760,000; such as from 50,000 Da to 740,000;for example from 50,000 Da to 720,000; such as from 50,000 Da to700,000; for example from 50,000 Da to 680,000; such as from 50,000 Dato 660,000; for example from 50,000 Da to 640,000; such as from 50,000Da to 620,000; for example from 50,000 Da to 600,000; such as from50,000 Da to 580,000; for example from 50,000 Da to 560,000; such asfrom 50,000 Da to 540,000; for example from 50,000 Da to 520,000; suchas from 50,000 Da to 500,000; for example from 50,000 Da to 480,000;such as from 50,000 Da to 460,000; for example from 50,000 Da to440,000; such as from 50,000 Da to 420,000; for example from 50,000 Dato 400,000; such as from 50,000 Da to 380,000; for example from 50,000Da to 360,000; such as from 50,000 Da to 340,000; for example from50,000 Da to 320,000; such as from 50,000 Da to 300,000; for examplefrom 50,000 Da to 280,000; such as from 50,000 Da to 260,000; forexample from 50,000 Da to 240,000; such as from 50,000 Da to 220,000;for example from 50,000 Da to 200,000; such as from 50,000 Da to180,000; for example from 50,000 Da to 160,000; such as from 50,000 Dato 140,000; for example from 50,000 Da to 120,000; such as from 50,000Da to 100,000; for example from 50,000 Da to 80,000; such as from 50,000Da to 60,000; for example from 100,000 Da to 1,000,000; such as from100,000 Da to 980,000; for example from 100,000 Da to 960,000; such asfrom 100,000 Da to 940,000; for example from 100,000 Da to 920,000; suchas from 100,000 Da to 900,000; for example from 100,000 Da to 880,000;such as from 100,000 Da to 860,000; for example from 100,000 Da to840,000; such as from 100,000 Da to 820,000; for example from 100,000 Dato 800,000; such as from 100,000 Da to 780,000; for example from 100,000Da to 760,000; such as from 100,000 Da to 740,000; for example from100,000 Da to 720,000; such as from 100,000 Da to 700,000; for examplefrom 100,000 Da to 680,000; such as from 100,000 Da to 660,000; forexample from 100,000 Da to 640,000; such as from 100,000 Da to 620,000;for example from 100,000 Da to 600,000; such as from 100,000 Da to580,000; for example from 100,000 Da to 560,000; such as from 100,000 Dato 540,000; for example from 100,000 Da to 520,000; such as from 100,000Da to 500,000; for example from 100,000 Da to 480,000; such as from100,000 Da to 460,000; for example from 100,000 Da to 440,000; such asfrom 100,000 Da to 420,000; for example from 100,000 Da to 400,000; suchas from 100,000 Da to 380,000; for example from 100,000 Da to 360,000;such as from 100,000 Da to 340,000; for example from 100,000 Da to320,000; such as from 100,000 Da to 300,000; for example from 100,000 Dato 280,000; such as from 100,000 Da to 260,000; for example from 100,000Da to 240,000; such as from 100,000 Da to 220,000; for example from100,000 Da to 200,000; such as from 100,000 Da to 180,000; for examplefrom 100,000 Da to 160,000; such as from 100,000 Da to 140,000; forexample from 100,000 Da to 120,000; such as from 150,000 Da to1,000,000; for example from 150,000 Da to 960,000; such as from 150,000Da to 940,000; for example from 150,000 Da to 920,000; such as from150,000 Da to 900,000; for example from 150,000 Da to 880,000; such asfrom 150,000 Da to 860,000; for example from 150,000 Da to 840,000; suchas from 150,000 Da to 820,000; for example from 150,000 Da to 800,000;such as from 150,000 Da to 780,000; for example from 150,000 Da to760,000; such as from 150,000 Da to 740,000; for example from 150,000 Dato 720,000; such as from 150,000 Da to 700,000; for example from 150,000Da to 680,000; such as from 150,000 Da to 660,000; for example from150,000 Da to 640,000; such as from 150,000 Da to 620,000; for examplefrom 150,000 Da to 600,000; such as from 150,000 Da to 580,000; forexample from 150,000 Da to 560,000; such as from 150,000 Da to 540,000;for example from 150,000 Da to 520,000; such as from 150,000 Da to500,000; for example from 150,000 Da to 480,000; such as from 150,000 Dato 460,000; for example from 150,000 Da to 440,000; such as from 150,000Da to 420,000; for example from 150,000 Da to 400,000; such as from150,000 Da to 380,000; for example from 150,000 Da to 360,000; such asfrom 150,000 Da to 340,000; for example from 150,000 Da to 320,000; suchas from 150,000 Da to 300,000; for example from 150,000 Da to 280,000;such as from 150,000 Da to 260,000; for example from 150,000 Da to240,000; such as from 150,000 Da to 220,000; for example from 150,000 Dato 200,000; such as from 150,000 Da to 180,000; for example from 150,000Da to 160,000.

Connector

In one embodiment a number of binding molecules (BM) associate with amultimerization domain to form a binding molecule multimer (BMmultimer). The association may be direct—between the binding moleculeand the multimerization domain, or may be via an association with aconnector, such as a connector molecule, which interconnects the bindingmolecule and the multimerization domain. A multimerization domain with aconnector is an example of linkers according to the present invention,

In another embodiment the linker of the detection molecule comprises orconsists of one or more connectors and/or connector molecules.Connectors may be formed of chemical bonds and/or molecules.

The attachment of binding molecules to the multimerization domain mayinvolve covalent or non-covalent connectors, and may involve smallreactive groups as well as large protein-protein interactions. Thecoupling of multimerization domains and binding molecules involve theassociation of an entity X (attached to or part of the multimerizationdomain) and an entity Y (attached to or part of the binding moleculeand/or label). Thus, the connector that connects the multimerizationdomain and the binding molecule comprises an XY portion.

A connector according to the invention in on one embodiment comprisesone or more chemical bond formed between the linker/multimerizationdomain, and each of the binding molecules and labels.

A connector according to the invention in on one embodiment comprisesone or more connector molecules associated with thelinker/multimerization domain, and each of the binding molecules andlabels.

In one embodiment, a number of MHC complexes associate with amultimerization domain to form a MHC multimer. The attachment of MHCcomplexes to the multimerization domain may involve covalent ornon-covalent connectors, and may involve small reactive groups as wellas large protein-protein interactions.

The coupling of multimerization domains and MHC complexes involve theassociation of an entity X (attached to or part of the multimerizationdomain) and an entity Y (attached to or part of the MHC complex and/orlabel). Thus, the connector that connects the multimerization domain andthe MHC complex comprises an XY portion.

-   -   Covalent connector. The XY linkage can be covalent, in which        case X and Y are reactive groups. In this case, X can be a        nucleophilic group (such as —NH₂, —OH, —SH, —NH—NH₂), and Y an        electrophilic group (such as CHO, COOH, CO) that react to form a        covalent bond XY; or Y can be a nucleophilic group and X an        electrophilic group that react to form a covalent bond XY. Other        possibilities exist, e.g either of the reactive groups can be a        radical, capable of reacting with the other reactive group.    -   X and Y can be reactive groups naturally comprised within the        multimerization domain and/or the binding molecule such as MHC        complex, or they can be artificially added reactive groups.        Thus, connectors containing reactive groups can be linked to        either of the multimerization domain and binding molecule such        as MHC complex; subsequently the introduced reactive group(s)        can be used to covalently link the multimerization domain and        binding molecule such as MHC complex.    -   Example natural reactive groups of binding molecule such as MHC        complexes include amino acid side chains comprising —NH₂, —OH,        —SH, and —NH—. Example natural reactive groups of        multimerization domains include hydroxyls of polysaccharides        such as dextrans, but also include amino acid side chains        comprising —NH₂, —OH, —SH, and —NH— of polypeptides, when the        polypeptide is used as a multimerization domain.    -   In some MHC multimers, one of the polypeptides of the MHC        complex (i.e. the β2M, heavy chain or the antigenic peptide) is        linked by a protein fusion to the multimerization domain. Thus,        during the translation of the fusion protein, an acyl group        (reactive group X or Y) and an amino group (reactive group Y        or X) react to form an amide bond. Example MHC multimers where        the bond between the multimerization domain and the MHC complex        is covalent and results from reaction between natural reactive        groups, include MHC-pentamers (described in US        patent 2004209295) and MHC-dimers, where the linkage between        multimerization domain and MHC complex is in both cases        generated during the translation of the fusion protein.    -   Example artificial reactive groups include reactive groups that        are attached to the multimerization domain or the binding        molecule such as MHC complex, through association of a connector        molecule comprising the reactive group. The activation of        dextran by reaction of the dextran hydroxyls with divinyl        sulfone, introduces a reactive vinyl group that can react with        e.g. amines of the binding molecule such as MHC complex, to form        an amine that now links the multimerization domain (the dextran        polymer) and the binding molecule such as MHC complex. An        alternative activation of the dextran multimerization domain        involves a multistep reaction that results in the decoration of        the dextran with maleimide groups, as described in the patent        Siiman et al. U.S. Pat. No. 6,387,622. In this approach, the        amino groups of MHC complexes are converted to —SH groups,        capable of reacting with the maleimide groups of the activated        dextran. Thus, in the latter example, both the reactive group of        the multimerization domain (the maleimide) and the reactive        group of the MHC complex (the thiol) are artificially        introduced.    -   Sometimes activating reagents are used in order to make the        reactive groups more reactive. For example, acids such as        glutamate or aspartate can be converted to activated esters by        addition of e.g. carbodiimid and NHS or nitrophenol, or by        converting the acid moiety to a tosyl-activated ester. The        activated ester reacts efficiently with a nucleophile such as        —NH₂, —SH, —OH, etc.

For the purpose of this invention, the multimerization domains(including small organic scaffold molecules, proteins, proteincomplexes, polymers, beads, liposomes, micelles, cells) that form acovalent bond with the binding molecules such as MHC complexes can bedivided into separate groups, depending on the nature of the reactivegroup that the multimerization domain contains. One group comprisemultimerization domains that carry nucleophilic groups (e.g. —NH₂, —OH,—SH, —CN, —NH—NH₂), exemplified by polysaccharides, polypeptidescontaining e.g. lysine, serine, and cysteine; another group ofmultimerization domains carry electrophilic groups (e.g. —COOH, —CHO,—CO, NHS-ester, tosyl-activated ester, and other activated esters,acid-anhydrides), exemplified by polypeptides containing e.g. glutamateand aspartate, or vinyl sulfone activated dextran; yet another group ofmultimerization domains carry radicals or conjugated double bonds.

-   -   Likewise, binding molecules such as MHC complexes can be divided        into separate groups, depending on the nature of the reactive        group comprised within the binding molecule. One group comprise        binding molecules such as MHCs that carry nucleophilic groups        (e.g. —NH₂, —OH, —SH, —CN, —NH—NH₂), e.g. lysine, serine, and        cysteine; another group of binding molecules such as MHCs carry        electrophilic groups (e.g. —COOH, —CHO, —CO, NHS-ester,        tosyl-activated ester, and other activated esters,        acid-anhydrides), exemplified by e.g. glutamate and aspartate;        yet another group of binding molecules such as MHCs carry        radicals or conjugated double bonds.    -   The reactive groups of the binding molecule such as MHC complex        are either carried by the amino acids of the binding molecule        such as MHC-peptide complex (and may be comprised by any of the        peptides of the MHC-peptide complex, including the antigenic        peptide), or alternatively, the reactive group of the binding        molecule such as MHC complex has been introduced by covalent or        non-covalent attachment of a molecule containing the appropriate        reactive group.    -   Preferred reactive groups in this regard include —CSO₂OH,        phenylchloride, —SH, —SS, aldehydes, hydroxyls, isocyanate,        thiols, amines, esters, thioesters, carboxylic acids, triple        bonds, double bonds, ethers, acid chlorides, phosphates,        imidazoles, halogenated aromatic rings, any precursors thereof,        or any protected reactive groups, and many others.    -   Reactions that may be employed include acylation (formation of        amide, pyrazolone, isoxazolone, pyrimidine, comarine,        quinolinon, phthalhydrazide, diketopiperazine, benzodiazepinone,        and hydantoin), alkylation, vinylation, disulfide formation,        Wittig reaction, Horner-Wittig-Emmans reaction, arylation        (formation of biaryl or vinylarene), condensation reactions,        cycloadditions ((2+4), (3+2)), addition to carbon-carbon        multiplebonds, cycloaddition to multiple bonds, addition to        carbon-hetero multiple bonds, nucleophilic aromatic        substitution, transition metal catalyzed reactions, and may        involve formation of ethers, thioethers, secondary amines,        tertiary amines, beta-hydroxy ethers, beta-hydroxy thioethers,        beta-hydroxy amines, beta-amino ethers, amides, thioamides,        oximes, sulfonamides, di- and tri-functional compounds,        substituted aromatic compounds, vinyl substituted aromatic        compounds, alkyn substituted aromatic compounds, biaryl        compounds, hydrazines, hydroxylamine ethers, substituted        cycloalkenes, substituted cyclodienes, substituted 1, 2, 3        triazoles, substituted cycloalkenes, beta-hydroxy ketones,        beta-hydroxy aldehydes, vinyl ketones, vinyl aldehydes,        substituted alkenes, substituted alkenes, substituted amines,        and many others.    -   Binding molecule dextramers, preferably MHC dextramers, can be        made by covalent coupling of binding molecules such as MHC        complexes to the dextran backbone, e.g. by chemical coupling of        binding molecules such as MHC complexes to dextran backbones.    -   The MHC complexes can be coupled through either heavy chain or        β2-microglobulin if the MHC complexes are MHC I or through        α-chain or β-chain if the MHC complexes are MHC II. MHC        complexes can be coupled as folded complexes comprising heavy        chain/beta2microglobulin or α-chain/β-chain or either        combination together with peptide in the peptide-binding cleft.        Alternatively either of the protein chains can be coupled to        dextran and then folded in vitro together with the other chain        of the MHC complex not coupled to dextran and together with        peptide.    -   Direct coupling of binding molecules such as MHC complexes to        dextran multimerization domain can be via an amino group or via        a sulphide group. Either group can be a natural component of the        binding molecule such as MHC complex or attached to the binding        molecules such as MHC complex chemically. Alternatively, a        cysteine may be introduced into the genes of the binding        molecules such as either chain of the MHC complex.    -   Another way to covalently link binding molecules such as MHC        complexes to dextran multimerization domains is to use the        antigenic peptide as a connector between MHC and dextran.        Connectors containing antigenic peptide at one end is coupled to        dextran. Antigenic peptide here means a peptide able to bind MHC        complexes in the peptide-binding cleft. As an example, 10 or        more antigenic peptides may be coupled to one dextran molecule.        When MHC complexes are added to such peptide-dextran construct        the MHC complexes will bind the antigenic peptides and thereby        MHC-peptide complexes are displayed around the dextran        multimerization domain. The antigenic peptides can be identical        or different from each other. Similarly MHC complexes can be        either identical or different from each other as long as they        are capable of binding one or more of the peptides on the        dextran multimerization domain.    -   Non-covalent connector. The linkage molecule that connects the        multimerization domain and the binding molecule such as MHC        complex comprises an XY portion. Above different kinds of        covalent linkages XY were described. However, the XY linkage can        also be non-covalent. Non-covalent XY linkages can comprise        natural dimerization pairs such as antigen-antibody pairs,        DNA-DNA interactions, or can include natural interactions        between small molecules and proteins, e.g. between biotin and        streptavidin. Artificial XY examples include XY pairs such as        His₆ tag (X) interacting with Ni-NTA (Y) and PNA-PNA        interactions.    -   Protein-protein interactions. The non-covalent connector may        comprise a complex of two or more polypeptides or proteins, held        together by non-covalent interactions. Example polypeptides and        proteins belonging to this group include Fos/Jun, Acid/Base        coiled coil structure, antibody/antigen (where the antigen is a        peptide), and many others.    -   A preferred embodiment involving non-covalent interactions        between polypeptides and/or proteins are represented by the        Pentamer structure described in US patent 2004209295.    -   Another preferred embodiment involves the use of antibodies,        with affinity for the surface of the binding molecule (BM).        Thus, an anti-BM antibody, with its two binding sites, will bind        two binding molecules and in this way generate a bivalent BM        multimer. In addition, the antibody can stabilize the BM through        the binding interactions.    -   Another preferred embodiment involves the use of antibodies,        with affinity for the surface of MHC opposite to the        peptide-binding groove. Thus, an anti-MHC antibody, with its two        binding sites, will bind two MHC complexes and in this way        generate a bivalent MHC multimer. In addition, the antibody can        stabilize the MHC complex through the binding interactions. This        is particularly relevant for MHC class II complexes, as these        are less stable than class I MHC complexes.    -   Polynucleotide-polynucleotide interactions. The non-covalent        connector may comprise nucleotides that interact non-covalently.        Example interactions include PNA/PNA, DNA/DNA, RNA/RNA, LNA/DNA,        and any other nucleic acid duplex structure, and any combination        of such natural and unnatural polynucleotides such as DNA/PNA,        RNA/DNA, and PNA/LNA.    -   Protein-small molecule interactions. The non-covalent connector        may comprise a macromolecule (e.g. protein, polynucleotide) and        a small molecule ligand of the macromolecule. The interaction        may be natural (i.e., found in Nature, such as the        Streptavidin/biotin interaction) or non-natural (e.g. His-tag        peptide/Ni-NTA interaction). Example interactions include        Streptavidin/biotin and anti-biotin antibody/biotin.    -   Combinations—non-covalent connector molecules. Other        combinations of proteins, polynucleotides, small organic        molecules, and other molecules, may be used to link the MHC to        the multimerization domain. These other combinations include        protein-DNA interactions (e.g. DNA binding protein such as the        gene regulatory protein CRP interacting with its DNA recognition        sequence), RNA aptamer-protein interactions (e.g. RNA aptamer        specific for growth hormone interacting with growth hormone)    -   Synthetic molecule-synthetic molecule interaction. The        non-covalent connector may comprise a complex of two or more        organic molecules, held together by non-covalent interactions.        Example interactions are two chelate molecules binding to the        same metal ion (e.g. EDTA-Ni⁺⁺-NTA), or a short polyhistidine        peptide (e.g. His₆) bound to NTA-Ni⁺⁺.

In another preferred embodiment the multimerization domain is a bead.The bead is covalently or non-covalently coated with binding molecules,such as BM multimers or BM monomers, for example MHC multimers or singleMHC complexes, through non-cleavable or cleavable connectors. As anexample, the bead can be coated with streptavidin monomers, which inturn are associated with biotinylated binding molecules such as MHCcomplexes; or the bead can be coated with streptavidin tetramers, eachof which are associated with 0, 1, 2, 3, or 4 biotinylated bindingmolecules such as MHC complexes; or the bead can be coated withBM-dextramers such as MHC-dextramers where e.g. the reactive groups ofthe BM- or MHC-dextramer (e.g. the divinyl sulfone-activated dextranbackbone) has reacted with nucleophilic groups on the bead, to form acovalent linkage between the dextran of the dextramer and the beads.

In another preferred embodiment, the binding molecule multimers such asMHC multimers described above (e.g. where the multimerization domain isa bead) further contains a flexible or rigid, and water soluble,connector that allows for the immobilized binding molecules such as MHCcomplexes to interact efficiently with cells; such as T-cells withaffinity for the MHC complexes. In yet another embodiment, the connectoris cleavable, allowing for release of the binding molecules such as MHCcomplexes from the bead. If T-cells have been immobilized, by binding tothe BMs such as MHC complexes, the T-cells can very gently be releasedby cleavage of this cleavable connector. Most preferably, the connectoris cleaved at physiological conditions, allowing for the integrity ofthe isolated cells.

Further examples of connector molecules that may be employed in thepresent invention include Calmodulin-binding peptide (CBP), 6×HIS,Protein A, Protein G, biotin, Avidine, Streptavidine, Strep-tag,Cellulose Binding Domain, Maltose Binding Protein, S-Peptide Tag, ChitinBinding Tag, Immuno-reactive Epitopes, Epitope Tags, GST taggedproteins, E2Tag, HA Epitope Tag, Myc Epitope, FLAG Epitope, AU1 and AU5Epitopes, Glu-Glu Epitope, KT3 Epitope, IRS Epitope, Btag Epitope,Protein Kinase-C Epitope, VSV Epitope.

The list of dimerization- and multimerization domains, describedelsewhere in this document, define alternative non-covalent connectorsbetween the multimerization domain and the binding molecule such as theMHC complex.

The abovementioned dimerization- and multimerization domains representspecific binding interactions. Another type of non-covalent interactionsinvolves the non-specific adsorption of e.g. proteins onto surfaces. Asan example, the non-covalent adsorption of proteins onto glass beadsrepresents this class of XY interactions. Likewise, the interaction ofbinding molecules such as MHC complexes (comprising full-lengthpolypeptide chains, including the transmembrane portion) with the cellmembrane of for example dendritic cells is an example of a non-covalent,primarily non-specific XY interaction.

In some of the abovementioned embodiments, several multimerizationdomains (e.g. streptavidin tetramers bound to biotinylated MHCcomplexes) are linked to another multimerization domain (e.g. the bead).For the purpose of this invention we shall call both the smaller and thebigger multimerization domain, as well as the combined multimerizationdomain, for multimerization domain.

Kit of Parts

The composition of the invention may form part of a kit. Thus, yet anaspect of the invention relates to a kit of parts comprising

-   -   a. one or more detection molecules,wherein each detection        molecule or set of detection molecules are as disclosed herein        throughout, and    -   b. one or more additional components.

In one embodiment the kit of parts comprises

-   -   a composition comprising a detection molecule, wherein said        detection molecule comprises a DNA molecule label, according to        the invention; and    -   one or more sets of primers for amplifying the nucleic acid        molecule label, such as DNA molecule label.

In one embodiment said additional components comprise reagents fordetecting and/or amplifying the label of the detection molecule.

In one embodiment said additional components comprise reagents fordetecting and/or amplifying the nucleic acid label of the detectionmolecule.

In one embodiment said additional components comprise one or more primersets capable of amplifying the label of the nucleic acid label, such asamplifying the barcode region of the nucleic acid label viahybridization to the primer regions of the nucleic acid label.

Detection Method

It is an aspect of the invention to provide a detection methodcomprising one or more steps of:

-   -   a. Combining a sample with at least one detection molecule;        wherein the detection molecule comprises a binding molecule        (BM), a linker (Li), and a label (La), each as defined according        to the present invention; and wherein said sample comprises at        least one cell and/or entity,    -   b. Incubating the at least one detection molecule and the        sample;    -   c. Isolating and/or detecting the at least one detection        molecule of step b), and    -   d. Optionally determining the identity of the at least one        detection molecule of step c).

It is understood that each step of the detection method, namely thesample, the isolation and/or detection and the determination of label,individually can be selected from any of the samples, isolation and/ordetection steps and determination of label steps disclosed hereinthroughout. Thus, any combination of sample, isolation and/or detectionand determination of label are encompassed within the present disclosureand invention.

In one embodiment there is provided a method comprising the followingsteps:

-   -   a. Combining at least one cell with at least one detection        molecule, wherein the detection molecule comprises a binding        molecule (BM), a linker (Li), and a label (La);    -   b. Allowing the detection molecules to recognize and bind cells        through their binding molecule (BM);    -   c. Detecting and/or isolating cell-detection molecule complexes        formed in step (b); and    -   d. Identifying detection molecules capable of binding to a cell        in step (b)

Also provided is a method for detecting antigen-responsive cells in asample comprising:

-   -   a. providing one or more multimeric major histocompatibility        complexes (MHC's) according to the invention or a composition        according to the invention;    -   b. contacting said multimeric MHC's with said sample; and    -   c. detecting binding of the multimeric MHC's to said antigen        responsive cells, thereby detecting cells responsive to an        antigen present in a set of MHC's; wherein said binding is        detected by amplifying the barcode region of said nucleic acid        molecule linked to the one or more MHC's.

It is understood that the detection molecule and each of the componentsof the detection molecule (BM, Li and La) referred to in the detectionmethods of the invention can individual be as outlined herein elsewhere.

The detection molecules and methods (e.g. detection methods) of thepresent invention can be used for or applied in a number of methods andassays. The assays for which the detection molecules and the methods ofusing same can be employed include assays for recognizing or detectingwell-known targets or epitopes (such as diagnosis, detection ofantigen-specific T-cells), and assays for investigating novel or unknowntargets or epitopes (such as epitope discovery, whereby the bindingproperties of the detection molecule are unknown).

Methods include, but are not limited to one or more of: epitopediscovery, analytical studies, diagnosis, therapy/treatment, detectionof antigen-responsive cells, detection of antigen-specific T-cells,characterization of cells of their detection molecule-bindingproperties, T-cell epitope mapping, immune-related therapies,immune-recognition discovery and measuring immune reactivity aftervaccination.

In one embodiment the present invention allows for detection of multipleantigen-responsive cells present in the same sample, wherein saidantigen-responsive cells have different specificities and detectdifferent antigens and/or epitopes. This can be achieved since eachdetection molecule has a unique and detectable label (La) associatedwith a specific binding molecule (BM) for the antigen-responsive cell.

In one embodiment the detection method further comprises one or moresteps of providing a sample, preferably a sample comprising at least oneentity and/or at least one cell.

In one embodiment the detection method further comprises one or moresteps of pre-treatment of the sample, and/or pre-treatment of cells ofthe sample.

The method thus in one embodiment involves first the mixing of one ormore detection molecules capable of binding certain cells with a samplecomprising cells, followed by complex formation between some or all ofthe detection molecules and some or all of the cells, and finallydetection and/or isolation of the cell-detection molecule complexesformed.

It follows that in step b) the one or more detection molecules areallowed to associate with, recognize, and/or bind to said at least onecell and/or entity through their binding molecule.

The detection method in some embodiment further comprises one or moresteps of separating unbound detection molecules from cell- orentity-detection molecule complexes.

The detection method in some embodiment further comprises one or moresteps of removing unbound detection molecules by washing and/orcentrifuging.

In one embodiment in step c) and d) said detection molecule is comprisedin a cell-detection molecule complex or an entity-detection moleculecomplex.

In another embodiment in step c) and d) said detection molecule is notcomprised in a cell-detection molecule complex or an entity-detectionmolecule complex.

In one embodiment in step c) and d) said detection molecule is no longercomprised in a cell-detection molecule complex or an entity-detectionmolecule complex, wherein said detection molecule has previouslyinteracted with a cell-detection molecule complex or an entity-detectionmolecule complex.

In steps c) and/or d) of the detection method the sorting and/ordetecting thus ensues on the detection molecule per se, or thecell-detection molecule complex (or entity-detection molecule complex).

In one embodiment step c) comprises isolating and detecting the at leastone detection molecule.

In one embodiment step c) comprises first isolating and then detectingthe at least one detection molecule.

In one embodiment step c) comprises detecting the at least one detectionmolecule.

In one embodiment step c) comprises isolating the at least one detectionmolecule.

In one embodiment step c) comprises first detecting and then isolatingthe at least one detection molecule.

The isolation in step c) may be performed by any method known to theskilled person. Isolation is used interchangeably with enrichment andcell sorting herein.

In a particular embodiment the detection molecule further comprises oneor more fluorescent labels, which are useful in the sorting of specificcell populations. Examples of specific cell populations include T-cells(CD8 or CD4 restricted), other immune cells or specifically MHC multimerbinding T-cells

In one embodiment, specific cell populations are sorted by methodscomprising one or more of flow cytometry, FACS sorting, centrifugation,washing, precipitation, filtration and affinity column sorting, or anyother means of cell sorting/selection.

In one embodiment in step c) isolating comprises sorting of cellpopulations based on the functional response to a stimuli (responsive ornon-responsive population), such as cytokine secretion, phosphorylation,calcium release.

In one embodiment in step c) isolating comprises sorting of cellpopulations based on phenotype, such as by linking a certain set ofphenotypic characteristics to the antigen-responsiveness.

In one embodiment in step c) isolating comprises immobilization of thedetection molecule and/or cell-detection molecule complexes, such as byprecipitating cells, such as by centrifugation, by immunoprecipitation,or any other means that precipitates the cells. or by binding thecell-detection molecule complexes to a bead, a particle, anothersurface, an antibody or an MHC complex.

In one embodiment said immobilization of the detection molecule and/orcell-detection molecule complexes comprises hybridization onto an array.

In one embodiment said immobilization of the detection molecule and/orcell-detection molecule complexes by hybridization onto an arraycomprises a nucleic acid/nucleic acid-interaction between the nucleicacid label of the detection molecule and an antisense nucleic acidsequence in the array.

In one embodiment said immobilization of the detection molecule and/orcell-detection molecule complexes by hybridization onto an arraycomprises a DNA/DNA-interaction between the DNA label of the detectionmolecule and an antisense DNA in the array.

In one embodiment said detecting in step c) and/or determining theidentity in step d) comprises one or more steps of adding primaryantibodies that bind to the immobilized detection molecule and/orcell-detection molecule complexes and detecting said primary antibodiesdirectly wherein the primary antibody is labelled, or indirectly byadding labelled secondary antibodies.

In one embodiment said detecting in step c) and/or determining theidentity in step d) comprises one or more steps of detecting theimmobilized detection molecule and/or cell-detection molecule complexesby monitoring read-out from a second label such as a fluorophore.

In one embodiment detecting in step c) and/or determining the identityin step d) comprises interaction between ‘coating DNA’ on the cellsurface and the DNA label of the detection molecule.

In one embodiment detecting in step c) and/or determining the identityin step d) comprises protease cleavage of the peptide label of thedetection molecule.

In one embodiment detecting in step c) and/or determining the identityin step d) comprises transfer of a cell surface moiety to the detectionmolecule (e.g. a ‘peptide tag’).

In one embodiment detecting in step c) and/or determining the identityin step d) comprises detection of the label based on the physicalcharacteristics of the label, including mass, sequence, charge, volume,size, dimensions, fluorescence, absorption, emission, NMR spectra andothers.

In one embodiment detecting in step c) and/or determining the identityin step d) comprises amplification of the label.

In one embodiment detecting in step c) and/or determining the identityin step d) comprises sequencing of the label (e.g. DNA sequencing,peptide sequencing).

In one embodiment detecting in step c) and/or determining the identityin step d) comprises amplification of the barcode sequence of a nucleicacid label by PCR and/or sequencing of the barcode sequence.

In one embodiment sequencing comprises deep sequencing or nextgeneration sequencing.

In one embodiment detecting in step c) and/or determining the identityin step d) comprises mass spectrometry.

In one embodiment detecting in step c) and/or determining the identityin step d) comprises one or more of gel electrophoresis, gel filtration,PAGE, column fractionation, PCR and QPCR.

The detection method in some embodiments further comprises one or moresteps of single-cell sorting and sequencing, such as single-cell T cellsorting of and single-cell TCR sequencing.

In an aspect of the present invention a nucleic acid label comprising abarcode will serve as a specific label for a given binding molecule,such as a peptide-MHC molecule that is multimerized to form a MHCmultimer. The multimer can be composed of MHC class I, class II, CD1 orother MHC-like molecules. Thus, when the term MHC multimers is used thisincludes all MHC-like molecules. The MHC multimer is formed throughmultimerization of peptide-MHC molecules via different backbones. Thebarcode will be co-attached to the multimer and serve as a specificlabel for a particular peptide-MHC complex. In this way up to 1000 to10.000 (or potentially even more) different peptide-MHC multimers can bemixed, allow specific interaction with T-cells from blood or otherbiological specimens, wash-out unbound MHC-multimers and determine thesequence of the DNA-barcodes. When selecting a cell population ofinterest, the sequence of barcodes present above background level, willprovide a fingerprint for identification of the antigen responsive cellspresent in the given cell-population. The number of sequence-reads foreach specific barcode will correlate with the frequency of specificT-cells, and the frequency can be estimated by comparing the frequencyof reads to the input-frequency of T-cells. This strategy may expand ourunderstanding of T-cell recognition.

In one embodiment a nucleic acid-label, DNA-label or DNA-barcode servesas a specific label for certain antigen specific T-cells and can be usedto determine the specificity of a T-cell after e.g. single-cell sorting,functional analyses or phenotypical assessments. In this way antigenspecificity can be linked to both the T-cell receptor sequence (that canbe revealed by single-cell sequencing methods) and functional andphenotypical characteristics of the antigen specific cells.

Furthermore, this strategy may allow for attachment of several different(sequence related) peptide-MHC multimers to a given T-cell—with thebinding avidity of the given peptide-MHC multimer determining therelative contribution of each peptide-MHC multimer to the binding ofcell-surface TCRs. By applying this feature it is possible to allow thedetermination of the fine-specificity/consensus recognition sequence ofa given TCR by use of overlapping peptide libraries or alaninesubstitution peptide libraries. Such determination is not possible withcurrent MHC multimer-based technologies.

As previously described a pool (library) of different sets of multimericmajor histocompatibility complexes (MHC's) may be used to analyze anoverall cell population for its specificity for peptides. Thus, anotheraspect of the invention relates to a composition comprising a subset ofmultimeric major histocompatibility complexes (MHC's) according to theinvention, wherein each set of MHC's has a different peptide, decisivefor T cell recognition and a unique “barcode” region in the DNAmolecule. In the present context, it is to be understood that eachspecific multimeric major histocompatibility complex is present in thecomposition with a certain number and that there is subset of differentmultimeric major histocompatibility complexes present in thecomposition.

Preferably all specific region for each multimeric MHC can be determinedwith only a few primer sets, preferably only one primer set. Thus, in anembodiment the primer regions in the DNA molecule are identical for eachset of MHC's. In this way only one primer set is required. In analternative embodiment, the mulitmeric MHC's are grouped by differentprimer sets, thereby allowing multiplication of different sets of themultimeric MHCs. In this way background noise may be limited, while alsoretrieving information of specific bindings. Thus, different primer setfor different sets of MHC's may be used.

The number of individual sets of multimeric MHC's may vary. Thus, in anembodiment the composition comprises at least 10 different sets ofmultimeric MHC's such as at least 100, such as at least 500, at least1000, at least 5000, such as in the range 10-50000, such as 10-1000 orsuch as 50-500 sets of MHC's.

Through analyses of barcode-sequence data, the antigen specificity ofcells in the specimen can be determined. When DNA-barcode #1 is detectedabove background level of reads it means that peptide-MHC multimer #1was preferentially bound to the selected cell type. Same goes forbarcode no. 2, 3, 4, 5, . . . etc. up to the potential combination ofmore than 1000 (but not restricted to this particular number). When thenumber of input cells are known, e.g. when cell populations of interestis captured via a fluorescence signal also attached to the multimer byflow cytometry-based sorting or other means of capturing/sorting, thespecific T-cell frequency can be calculated comparing the frequency ofbarcode-reads to the number of sorted T-cells.

In a particular embodiment the cells have not been permeabilized orlysed and therefore the detection molecules bind predominantly to theouter surface of the cells. In one embodiment the cells are intact, thecell membrane of the cells are intact.

In a particular embodiment the cells are permeable to the detectionmolecules wherefore the detection molecules bind to the outer surface ofthe cell as well as one or more components of the interior of the cell.

In a preferred embodiment, the combining of one or more cells (step a.),followed by incubation to allow binding (step b.), is performed in abuffer, such as a buffer selected from the group consisting of a PBSbuffer, a Tris buffer, a phosphate buffer and any other buffer withappropriate ionic strength and pH, suitable for formation of thecell-detection molecule complexes.

In a preferred embodiment the detecting of the cell-detection moleculecomplexes is done by first spinning down the cells (and the detectionmolecules attached to the cells), removing the supernatant, optionallywashing one or more times, and optionally amplifying the DNAoligonucleotide labels e.g. by PCR, then identifying the DNA label ofthe detection molecules still bound to the cells by e.g. sequencing ofthe DNA or by running polyacrylamide gel electrophoresis to identifydifferent DNA oligonucleotides based on their differential migration inthe gel, thereby identifying the detection molecules capable of bindingcells.

As known to the skilled person, unbound molecules should preferably beremoved. Thus, in an embodiment unbound (multimeric) MHC's are removedbefore amplification, e.g. by washing and/or spinning e.g. followed byremoving of the supernatant.

The detection of the barcode in one embodiment includes sequencing ofthe amplified barcode regions. Thus, in an embodiment the detection ofbarcode regions includes sequencing of said barcode region, such as bydeep sequencing or next generation sequencing.

In a preferred embodiment the isolation of cell-detection moleculecomplexes of step c. is done by immobilization of a population of cellsby binding to a marker molecule attached to a surface such as the bottomof a well of a microtiter plate, a bead such as a magnetic polystyrenebead, or a glass plate, where the marker molecule binds to all or asubset of the cells.

In a preferred embodiment the marker molecule is an antibody against theCD8 or CD4 receptor of T cells, and the binding molecule of thedetection molecule is a pMHC complex or a number of pMHC complexes,where the pMHC are of class I or class II, respectively.

In a preferred embodiment the cell-detection molecule complexes arefirst isolated, and then diluted and aliquoted into separate wells,under conditions where the majority of wells as a result will containzero or one cell, and wherefore the detection molecules bound to eachindividual cell may be determined.

It may also be advantageously to be able to sort the cells. Thus, in anembodiment the method further comprises cell sorting by e.g. flowcytometry such as FACS. This may e.g. be done if the backbone isequipped with a fluorescent marker. Thus, unbound cells may also beremoved/sorted.

In a preferred embodiment, the method is performed on known negativecontrol cell samples (i.e. cell samples that are known to not containcells that can bind to the detection molecules used in the method) toensure that non-specific (“faulty”) binding events occur. The methodwill typically be performed in parallel using two cell samples: one thatcontain cells that may comprise cells with binding affinity for one ormore of the detection molecules; and one cell sample that does notcontain cells with significant affinity for one or more detectionmolecules.

In a preferred embodiment, the method is performed including a negativecontrol detection molecule (i.e. a detection molecule that is expectedto not bind any of the cells).

In a preferred embodiment, the method is performed including a positivecontrol detection molecule (i.e. a detection molecule that is expectedto bind one or some of the cells).

As also known to the skilled person, the measured values are preferablycompared to a reference level. Thus, in an embodiment said bindingdetection includes comparing measured values to a reference level, e.g.a negative control and/or total level of response in the sample. In afurther embodiment, said amplification is PCR such as QPCR.

Isolating and/or Detecting Detection Molecules

Isolating detection molecules that are complexes with a certain cell, orthat have been in contact with a certain cell, can be done in many ways.

In one embodiment, the detection molecules that are in contact with thecells are enriched by removing detection molecules that are not incontact with cells. This can for example be done by centrifugation ofthe mixture of detection molecules and cell, whereby cells areprecipitated along with the detection molecules that bind them. When thesupernatant is then removed, the detection molecules that do not bindthe cells are removed. As a result, the detection molecules that bindthe cells are enriched for.

In one embodiment the incubation mixture of detection molecules andcells is centrifuged, in order to precipitate the cells, and thereby thedetection molecules bound to the cells. When the supernatant is theremoved, the detection molecules that did not bind to cells are removedas well. Optionally, one may repeat centrifugation and resuspension oneor more times, and then finally the enriched pool of detection moleculescan be examined, e.g. the identity and quantity of the recovereddetection molecules may be determined.

In one embodiment the incubation mixture of detection molecules andcells is centrifuged, in order to precipitate the cells, and thereby thedetection molecules bound to the cells. When the supernatant is thenremoved, the detection molecules that did not bind to cells are removedas well. Optionally, one may repeat centrifugation and resuspension oneor more times, and then finally the enriched pool of detection moleculescan be examined, e.g. the identity and quantity of the recovereddetection molecules may be determined. In one embodiment the resultingdetection molecules, still bound to cells, may be released from thecells, by e.g. mild acid or mild base treatment or protease treatment ifthe binding molecule or linker is a peptide. Once released, thedetection molecules may be examined by hybridization to an array ofanti-sense molecules. For example, if the labels of e.g. 100 detectionmolecules are oligonucleotide labels of different sequence, the arraycan comprise the corresponding 100 complementary sequences, positionedin a way so that a given position or area of the array comprises onlyoligonucleotides of a given sequence. The identity of theoligonucleotide label and hence the detection molecule to which it isbound, can then be determined when examining to which position in thearray that it anneals.

In another embodiment of the above embodiment, the cells and detectionmolecules are not separated before applying the cell-detection moleculecomplexes to the array. As a result, the cells will become immobilizedon the array at those positions where the sequence of theoligonucleotide label of the detection molecule bound to said cell iscomplementary to the oligonucleotide of the array. This way, both theidentity of the recovered detection molecules, as well as the number ofcells capable of binding said detection molecules, can be deduced fromthe array.

In the two embodiments immediately above, the binding of detectionmolecules to the array is detected for example by adding antibodies thatbind the binding molecule, and then staining these antibodies usingsecondary antibodies and e.g. chromophores or fluorophores as a readout,using standard procedures. Alternatively, the detection molecule cansimply carry a signal molecule, such as a fluorophore.

In one embodiment the incubation mixture of detection molecules andcells is centrifuged, under conditions and buffer choice where agradient develops in the centrifugation vial. As a result of thegradient being established in the centrifugation vial, the cells anddetection molecules and cell-detection molecule complexes will bedistributed along the gradient according to their weight and volume.Different cell types with different characteristics will locate todifferent positions in the gradient. Therefore, cells of differentkinds, along with the detection molecules that bind to them, can berecovered from specific positions along the gradient. Unbound detectionmolecules will locate far from the cells. After collecting the cells anddetection molecules bound to these cells, from a given position in thegradient, the identity and quantity of the recovered detection moleculesmay be determined.

In one embodiment the incubation mixture of detection molecules andcells is applied to a filter that retains structures of same or largersize than cells, and let smaller structures and molecules pass through.The retained cells may be resuspended and applied to the filter one ormore times. Finally, the identity and quantity of the recovereddetection molecules, i.e. the detection molecules that bound to thecells, may be determined.

In one embodiment the detection molecules are incubated with a solidsample comprising cells, such as a tissue section or biopsy. Afterincubation, the solid tissue or biopsy is washed one or more times, toremove unbound detection molecules. Finally, the remaining detectionmolecules, bound to cells of the solid tissue, can be identified andquantified by determining the identity and amount of label remaining onthe solid tissue.

In one embodiment, the detection molecules that are capable of bindingcertain cells are isolated by immobilizing said cells on e.g. beads, forexample coated with an antibody specific for said cells (e.g. beadscoated with anti-CD4 antibodies to specifically examine binding ofdetection molecules to CD4 T cells). When the cells have beenimmobilized on the beads, the beads are washed a couple of times toremove unbound or weakly bound detection molecules. The enriched pool ofdetection molecules, each of which are capable of binding theimmobilized cells, can then be identified and quantified byidentification and quantification of their respective labels.

In one embodiment, both the ability to secrete certain molecules, aswell as the ability to bind specific detection molecules, is used as ameans of enrichment of certain detection molecules. For example, ifcertain T cells of a cell sample are stimulated to secrete INF-γ (e.g byaddition of free peptides to the cell suspension, or by binding of MHCmultimers such as Dextramers or tetramers), and if bi-specificantibodies, recognizing both a T cell-specific protein on the cellsurface, and INF-γ, are added, the secreted INF-γ will be captured bythe bi-specific antibody and become immobilized on the cell surface. Ifthen beads, coated with anti-INF-γ antibody recognizing another face ofINF-γ are added, the cells that secrete INF-γ will become immobilized onthe beads. If then detection molecules comprising pMHC binding moleculesare added to the cell/bead suspension, detection molecules capable ofbinding T cells will become immobilized on the beads as well. Afterwashing to remove unbound detection molecules, the detection moleculesbinding to T cells that were secreting INF-γ, can be recovered. Finally,the identity and amount of the recovered detection molecules can bedetermined by identifying the label of the recovered detectionmolecules. Thus, in this approach, the detection molecules areidentified that bind to T cells, where said T cells are known to secreteINF-γ.

In one embodiment, the detection molecules carry both binding molecules(e.g. pMHC complexes) and anti-INF-γ antibody. INF-γ will be secretedfrom certain cells as a result of the stimulation brought about by thebinding of the detection molecule (i.e. brought about by the binding ofthe pMHC binding molecule), and the secreted INF-γ will be captured ondetection molecules bound to the same cell. These cells may beimmobilized on beads carrying anti-INF-γ antibodies. After washing toremove unbound detection molecules, the detection molecules binding to Tcells that were secreting INF-γ, can be recovered. Finally, the identityand amount of the recovered detection molecules can be determined byidentifying the label of the recovered detection molecules. Thus, inthis approach, the detection molecules are identified that bind to Tcells, where said T cells are known to secrete INF-γ.

In one embodiment, the detection molecules that are capable of bindingcertain cells are isolated by applying the incubation mixture of cellsand detection molecules to an affinity column, e.g. carrying antibodiesor other proteins that bind to a certain cell membrane protein of saidcells (where the cell membrane protein is indicative of the cell beinge.g. in a certain development stage, a certain stage in the mitoticcycle, or indicative of the cell being of a certain kind). It is ofcourse important that the column material allows the cells to migraterelatively unhindered through the column unless they bind to theaffinity target (e.g. antibody) on the column. The cells that bind tothe affinity column will migrate slower through the column and can berecovered from the later column fractions. The detection molecules thatdo not bind cells will flow through much faster than the cells, and canbe recovered in early fractions. The enriched pool of detectionmolecules, each of which are capable of binding the immobilized cells,can then be identified and quantified from the later fractions, byidentification and quantification of their respective labels.

In one embodiment, the detection molecules that are capable of bindingcertain cells are isolated by immobilizing all the detection moleculesrecovered after washing cells. Thus, in this embodiment, the detectionmolecules that are not bound to a cell are removed, by recovering thecell (e.g. by centrifugation, filtration through a filter that allowsmolecules but not cells to pass through, or any other means thatrecovers the cells (and the detection molecules bound to them) but doesnot recover the unbound detection molecules. The recovered cells may bere-suspended and the centrifugation/filtration process repeated, andthen the recovered cells with detection molecules bound are immobilizedon a surface (e.g. a microtiter-plate well coated with antibodies thatrecognize and bind detection molecules) or a bead (e.g. a bead coatedwith antibodies that recognize and bind detection molecules), or areprecipitated by adding primary antibodies that bind detection molecules,and secondary antibodies that bind the primary antibodies, thusresulting in the formation of large aggregates of cells, detectionmolecules, and primary and secondary antibodies, which can easily beprecipitated. Finally, the identity and quantity of each kind ofrecovered detection molecule is determined.

In one embodiment involving the isolation of detection molecules thatare capable of binding to certain cells, flow-activated cell sorting(FACS) is used. For example, the detection molecule may, in addition toa linker, binding molecule and label, comprise a fluorescent moiety(i.e. a fluorophore). Detection molecules and cells are incubated, andthose cells that are bound by a (fluorescent) detection molecule can beidentified and collected by a flow activated cell sorter (FACS). Thus,after sorting, the detection molecules that were capable of binding tocells, are collected together with the cells they bind to. The identityand amount of the recovered binding molecules can then be identified byidentification and quantification of the corresponding labels.

In one embodiment, the invention is used to perform single-cellphenotyping and/or analysis. Thus, most of the abovementioned isolationprocedures, in which (a subset of) cells are isolated, e.g. bycentrifugation, FACS sorting, or bead immobilization, the cells may(following their enrichment) be diluted and placed in separatecontainers (e.g. separate wells of a microtiter-plate well), at adilution where there is on average significantly less than one cell perwell. Consequently, those wells comprising one cell can be used to dosingle-cell phenotyping, by identifying the labels (and hence thedetection molecules) associated with the one cell present in the well.

In one embodiment, the binding molecule binds to an intracellulartarget, e.g. a protein or a mRNA. For such cases, the detection moleculeneeds to become intracellularized, either as a means of active orpassive transport across the fully functional membrane, or as a resultof the cells having been mildly permeabilized. The cells may bepermeabilized using standard methods such as those used routinely inintracellular staining (ICS) procedures.

Detecting detection molecules that are complexed with a certain cell, orthat have been in contact with a certain cell, can be done in many ways.

In one embodiment, the detection molecules that are or have been incontact with the cells are marked specifically. In one embodiment thedetection molecules carry DNA oligonucleotide labels (“label DNA”) ofdifferent lengths, i.e. the length of the DNA label identifies thedetection molecule. The surface of the sample cells is coated with DNAoligonucletides (“coating DNA”), for example by adding antibody-DNAconjugates where the antibody recognizes a particular cell surfacestructure.

The “label DNA” and “coating DNA” have complementary regions in their3′-ends but the 3′-end of “label DNA” is blocked and cannot serve asprimer for an extension reaction. When a detection molecule binds theDNA coated cells, the “label DNA” is brought into proximity of the“coating DNA”, allowing the complementary 3′ends to anneal. When apolymerase and radioactive nucleosides are added, the strandcomplementary to the “label DNA” is generated by extension from the3′end of “coating DNA”. DNA is then purified from the mixture andapplied to an appropriate gel, allowing separation of the radioactiveDNA fragments. The identity and amount of the labels (and hence thecorresponding detection molecules) can be determined from the positionand intensity of the band on the gel.

In one embodiment, the detection molecules that have been in contactwith cells are marked specifically. In one embodiment the detectionmolecules carry peptide labels. The peptide label consists of a codingregion and a protease cleavage site proximal to the linker that connectsthe label to the binding molecule. The surface of the sample cells iscoated with proteases capable of cleaving the peptide labels, forexample by adding antibody-protease conjugates where the antibodyrecognizes a particular cell surface structure. Thus, when the detectionmolecules bind to the sample cells, they are brought into proximity ofthe protease, which results in cleavage of the label and release of thecoding region from the detection molecule and hence, release from thecells. After centrifugation to precipitate the cells, the supernatantcan be analysed by mass spectrometry to determine the identity andamount of the labels that were released from the cells, and hence, theidentity and amount of detection molecules that bound to the cells inquestion.

In one embodiment, the detection molecules that have been in contactwith cells are marked, allowing their enrichment and analysis. Forexample, if the detection molecule carries—in addition to the bindingmolecule, linker and label—an enzyme catalyzing the transfer of a cellsurface moiety (e.g. a peptide fragment) from a cell surface protein tothe binding molecule of the detection molecule, when said surfaceprotein binds to the binding molecule. This peptide tag that has beenadded to the binding molecule, can then be used as a means of isolatingall the detection molecules that have been in contact with said cellsurface protein (e.g. by purifying the tagged detection molecules byapplying the incubation mixture to beads coated with anti-peptide tagantibodies). Finally, the isolated detection molecules can be identifiedby identification and quantification of their labels.

Determining the Identity of the Label

The identity of the label and thereby the identity of the bindingmolecule, may be determined from the label's physical characteristics,such as its mass, composition or sequence, charge, volume, size anddimensions, fluorescence, absorption, emission, NMR spectrum, and manyother physical characteristics.

The mass of a label can be determined by e.g. mass spectrometry.

The composition of a label can be determined by degradation to itscomponents (e.g a peptide being degraded to its amino acid residues),and the amounts of each of its components be determined by e.g. chemicalmethods (e.g. specific reactions), whereby the composition can bedetermined.

The sequence of a polymer can be determined by appropriate sequencingmethods. Thus, for peptides the sequence can be deduced by standardpeptide sequencing methods (e.g. Edman degradation). Likewise, fornatural oligonucleotides the sequence can be determined using standardsequencing reactions, involving chemical or enzymatic procedures.Oligonucleotides comprising certain unnatural nucleotides may also besequenced by some of these methodologies.

The sequence and amount of an oligonucleotide can also be determined bymeasuring its ability to anneal to complementary oligonucleotidesequences. This principle is applied in Q-PCR, where a polymerase chainreaction is performed. Here, forward and reverse primers are added tothe composition of labels, whereafter the amount corresponding(complementary) oligonucleotide template (label) can be determined bydetermining the amount of double-stranded PCR product generated atdifferent stages (number of cycles) of the PCR reaction.

Where a composition of detection molecules carry labels of similar sizeand/or mass, but different charge, these may be separated and identifiedand their relative amounts determined by e.g. running them on a gel. Ifthe labels are of similar size and dimension, and if the labels migratefrom the negative to positive electrode, the labels with the highestnegative charge will migrate the fastest through the gel. The relativeamounts of the different labels are reflected in the density, optionallystaining density if the gel is stained after electrophoresis, of thedifferent bands on the gel.

The volume, size and dimensions of a label molecule determines how fastit will flow through a column. For some column types, the largestmolecules flow through the column the fastest (e.g. gel filtrationcolumns). For other column types, the largest molecules flow through theslowest. Both column types can be used to separate and identify anddetermine the relative amounts of different labels, by applying thecomposition of labels to the top of the column, and measuring theflow-through time for each of the labels, as well as the amount of labeldetected in a given fraction. Labels emitting any kind of radiation canbe identified and quantified from its spectrum of emission and intensityof radiation. Detection of radiation is used in e.g. flow cytometers andspectrophotometers.

Labels can also be identified and quantified from their NMR signal.

Binding of Detection Molecules can be Detected Using Various Principles:

-   -   Fluidic samples are one embodiment of the present invention        where binding and detection of binding molecules can be used to        make a diagnostic analysis. One or more defined structures may        be measured.    -   Often entities like cells or other particles in a sample are        detected by binding molecules associated to surface receptors or        intracellular structures of the cell or binding structures        exposed on the bead

One way to analyse fluidic samples is using flow cytometry. In flowcytometry, the sample is a suspension of entities, which are moved toand centered in the flow cell (interrogation point) by co-flow withsheath fluid, or is directly injected into the instrument.

Liquid cell samples can be analyzed using a flow cytometer, able todetect and count individual cells passing in a stream through a laserbeam. For identification of specific cells in a sample, cells are taggedwith fluorescent labeled detection molecules by incubating cells withlabelled detection molecule and then force the cells with a large volumeof liquid through a nozzle creating a stream of individually spacedcells. Each cell passes through a laser beam and during the passage thelaser light is scattered and any fluorochrome bound to the cell isexcited and thereby fluoresce. Sensitive photomultipliers detect emittedfluorescence and thereby gives information of binding detectionmolecules to the surface of a given cell. By this method labelleddetection molecules can be used to identify specific cell populations incell samples of any individual. In here the term “labeling” of cellswith labelled detection molecules is used interchangeably with the term“staining”.

Flow cytometry allows detection of a single entity with a specified setof characteristics, within a population of entities with other sets ofcharacteristics. A major advantage of flow cytometry is that it allowsrapid analysis of multiple parameters for each individual entity,simultaneously.

Another example of a method measuring binding of labeled bindingmolecules (detection molecules) to structures in fluidic samples isdetection of cells or particles in the sample by binding labeled bindingmolecule followed by identification of labeled structures usingmicroscopy include light microscopy, immunofluorescence microscopy,confocal microscopy or other forms of microscopy. Basically the fluidicsample is stained with labeled binding molecule and non-binding bindingmolecule removed by washing. Then the sample is spread out on a slide orsimilar in a thin layer and labelled cells identified using amicroscope.

-   -   Measurement of detection molecule can also be used on solid        samples. Example solid sample include but is not limited to        solid tissue, blocks of solid tissue, slices of solid tissue,        cells or particles embedded in a solid matrix or any other solid        or semisolid sample.    -   Solid samples are typically analysed by placing them in        instruments as blocks or in the form of thin slide of material        on e.g. a glass plate. Solid material can then be labeled by        binding detection molecule and the amount of bound detection        molecule measured.

An example of a method measuring binding of detection molecule to solidsample is immunohistochemistry. This assay technique involvesimmobilization of the tissue slice on a glass slice, carrying the samplethrough the assay steps to the final analysis.

The means of detection typically involve photometric methods, microcopyand/or digital scanning of the sample. It may be simple light orfluorescence microscopy, for determination of chemiluminescence,morphology, shape, and fluorescence. Also, laser scanning techniques maybe employed, where confocal microscopy or standard light microscopy isemployed to give a 3- and 2-dimensional picture, respectively, of thesample. A digital image may be acquired, whereby the individual featurese.g. light intensity at a given area of the sample can be determined.

Another example of a method measuring binding of detection molecule tosolid sample is immunoelectron microscopy. In this technique detectionmolecules labeled with gold particle are applied to thin section ofsample which are then examined in a transmission electron microscope.This method is used to detect intracellular location of structures orparticular matter in a sample at high resolution.

Samples can also be attached to solid support and then incubated withdetection molecule and bound detection molecule measured. Alternativelydetection molecule of interest is bound to structure in sample beforeimmobilization of sample to solid support. This principle is especiallyuseful for binding detection molecules in solution to its bindingpartner in fluidic samples but can also be used on solid samples.

Below different principles for measurement of binding detection moleculeto sample immobilized to solid support is listed.

-   -   Molecule of interest is immobilized on solid support and        detected using labeled marker. Alternatively the sample is first        labeled and then immobilized on solid support. An example is        ELISA based and Radioimmuno based assays. In both assays the        immobilized sample is incubated with detection molecule (or the        sample is incubated with detection molecule and then immobilized        on solid support), then non-binding detection molecule is        removed by washing. Bound detection molecule is measured. The        detection molecule may be labeled directly or indirectly. In        ELISA the label is most often an enzyme while in radioimmuno        assays the label is a radioactive molecule. Other labeling        molecules may also be used like fluorochromes, chromophores or        other molecules that can be measured. Useful labeling molecules        are described elsewhere herein.    -   Structure of interest in sample is catched by its partner; the        specific detection molecule that is immobilized on solid        support. Bound sample or part of sample is then detected using a        labeled marker specific for the same or a different structure in        the bound sample.    -   Different detection molecules to two or more structures of        interest in same sample are immobilized on solid support in a        defined pattern. Structures of interest in sample are bound to        the defined areas of the support and are detected by a labeled        marker. Each individual structure binds to different partners        immobilized in different positions on the solid support. Thereby        the sample may be phenotyped.    -   Two or more solid supports (e.g. beads) with different        characteristics (e.g. size, fluorescence, fluorescence        intensities, labels), where each kind of bead has a specific        detection molecule immobilized. Structures of interest in sample        are bound to specific populations of beads, where each bead        population is defined by the detection molecule they have        immobilized. The different populations of beads are detected        through their special characteristics.

The sample analysed by the above described methods may first besubjected to another analysis e.g. separation according to size. Anexample is Blotting techniques were molecular structures of a sample arefirst separated according to size and then transferred to a solidsupport followed by detection with labeled detection molecule. Dependingon the structure to be analysed different forms of blotting exist, e.g.Western blotting for analysis of proteins, Northern blotting foranalysis of RNA and Southern blotting for analysis of DNA.

-   -   Binding may also be measured without using labeled detection        molecules for detection. A sample may be captured by immobilized        binding molecule, then eluted and quantified. Usually the        starting point is a sample in solution, such as a cell lysate,        growth medium or blood. The molecule or structure of interest        will have a well known and defined property which can be        exploited during the binding process. The process itself can be        thought of as an entrapment, with the target molecule becoming        trapped on a solid or stationary phase or medium. The other        molecules in solution will not become trapped as they do not        possess this property. The solid medium can then be removed from        the mixture, washed and the target molecule released from the        entrapment in a process known as elution.    -   Binding to the solid phase may be achieved by column        chromatography, whereby the solid medium is packed onto a        chromatography column, the initial mixture run through the        column to allow binding, a wash buffer run through the column        and the elution buffer subsequently applied to the column and        collected. These steps are usually done at ambient pressure (as        opposed to HPLC or FPLC).    -   Alternatively binding may be achieved using a batch treatment,        by adding the initial mixture to the solid phase in a vessel,        mixing, separating the solid phase (by centrifugation for        example), removing the liquid phase, washing, re-centrifuging,        adding the elution buffer, re-centrifuging and removing the        eluate.    -   Sometimes a hybrid method is employed, the binding is done by        the batch method, then the solid phase with the target molecule        bound is packed onto a column and washing and elution are done        on the column.    -   A third method, expanded bed adsorption, which combines the        advantages of the two methods mentioned above, has also been        developed. The solid phase particles are placed in a column        where liquid phase is pumped in from the bottom and exits at the        top. The gravity of the particles ensures that the solid phase        does not exit the column with the liquid phase.    -   Following elution the purified or enriched molecule or structure        can be quantitated, enriched further or used in new analytical        processes or treatment processes.    -   Measurement of alteration of physical state of sample or        structure in sample upon binding molecule. For example addition        of binding molecule to a fluidic sample may induce cross-linking        of structures, clumping and/or aggregation of sample. E.g.        antibodies or other binding molecules can bind large particles        and make the particle to clump or agglutinate. The antibody or        other molecule binds multiple particles and joins them, creating        a large complex.    -   Similar addition of binding molecule to solid sample may make        the solid sample becoming semi solid, fluidic or in another way        change texture.    -   Other physical characteristics may be changed as a consequence        of addition of binding molecule to sample and if measureable        they can be used for analysis of sample.    -   An example of a specific assay measuring alteration in the        physical state of a sample upon addition of binding molecule is        turbidimetry. This is a method for determining the concentration        of a substance in a solution by the degree of cloudiness or        turbidity it causes or by the degree of clarification it induces        in a turbid solution.    -   Indirect measurement of binding is measurement of the result of        the interaction between binding molecule and structure in sample        in contrast to direct measurement of the binding molecule bound        in sample. The result of interaction between binding molecule        and structure in sample can be measured in several ways indirect        ways.    -   Measurement of produced substance. Upon binding of binding        molecule to structure in sample the sample may release and/or        produce a substance that can be measured. Depending on the        nature of the sample different principles exists        -   The produced substance may be measured in solution either            directly or by detection with a labeled detection molecule.            The produced substance may be easily accessible for            detection or alternatively need to be extracted from sample            before measurement is possible.

One example of measurement of produced substance in solution isPolymerase Chain reaction (PCR). In this method fragments of DNA insolution are amplified by binding DNA primers (binding molecules) todefined areas of the DNA in sample. The amount of amplified DNA is thenmeasured.

Another example is measurement of produced soluble substance insidecells using intracellular flow cytometry. This can be done using blockof secretion of soluble substance (e.g. by monensin), permeabilizationof cell (by e.g. saponine) followed by immunofluorescent staining. Themethod involves the following steps: 1) An reagent able to blockextracellular secretion of cytokine is added, e.g. monensin thatinterrupt intracellular transport processes leading to accumulation ofproduced soluble factor, e.g. cytokine in the Golgi complex. 2) Fixationof cell membrane using mild fixator followed by permeabilization of cellmembrane by. e.g. saponine. 3) Addition of labelled detection moleculespecific for the produced soluble substance to be determined. 5)Measurement of labelled cells using a flow cytometer. Optionally thisanalysis can be combined with labeling with detection molecules specificfor surface exposed structures. If so these detection molecules areadded before step 2.

An alternative to this procedure is to trap secreted soluble factors onthe surface of the secreting cells followed by detection with specificdetection molecules as described by Manz, R. et al., Proc. Natl. Acad.Sci. USA 92:1921 (1995).

-   -   The produced substance may be immobilized to solid support        followed by detection using labeled marker. Principles for        immobilization and detection are as described elsewhere herein        for immobilization of samples and direct detection    -   An example of indirect detection of produced substance        immobilized on solid support is measurement of substances        secreted from stimulated cells by capture of the secreted        substance on solid support followed by detection with labeled        detection molecule. Secreted soluble substance in the        supernatant is immobilized on solid support either directly or        through a linker molecule. The cells may be stimulated by        addition of other cells to the sample, addition of antigens        (peptides/proteins) to the sample, addition of stimulatory        proteins or other molecules or stimulated by other means. The        amount of secreted substance can be measured in different ways:        -   Soluble substances secreted from individual cells can be            detected by capturing of the secreted soluble substances            locally by detection molecules, e.g. antibody specific for            the soluble substance. Soluble substance recognizing            detection molecules are then immobilised on a solid support            together with cells and soluble substances secreted by            individual cells are thereby captured in the proximity of            each cell. Bound soluble substance can be measured using            labelled detection molecule specific for the captured            soluble substance. The number of cells that has given rise            to labelled spots on solid support can then be enumerated            and these spots indicate the presence of specific cells that            have been stimulated with particular stimulator.        -   Soluble substances secreted from a population of cells are            detected by capture and detection of soluble substance            secreted from the entire population of specific cells. In            this case soluble substance do not have to be captured            locally close to each cell but the secreted soluble            substances may be captured and detected in the same well as            where the cells are or transferred to another solid support            with detection molecules for capture and detection e.g.            beads or wells of ELISA plate.        -   The produced substance is measured directly in solid sample            using principles as described for solid samples elsewhere            herein.    -   Measurement of effector function in sample. Binding of binding        molecule may also result in changes in effector function of        sample. Effector function is inhere any function of sample or        produced in sample. This type of measurement is only relevant        for samples comprising living cells, living organisms or other        living material. Examples of effector function of/in a sample        include but are not limited to cytolytic activity, catalytic        activity, ability to stimulate other cells or samples or ability        to induce apoptosis in sample itself or in other samples.

An example is measurement of activation of T cells in a sample bymeasurement of cytolytic activity of the T cell in a cytolytic assay,e.g. a chromium release assay.

-   -   Measurement of growth. Binding of binding molecule may induce or        inhibit growth in a sample. This type of measurement is only        relevant for samples comprising living cells, living organisms        or other living material. In cell samples growth can be measured        as proliferation of cells in the sample.    -   Examples of methods useful for measuring proliferation include        but are not limited to:        -   Detection of mRNA. Proliferation of T cells can be detected            by measurement of mRNA inside cell. Cell division and            proliferation requires production of new protein in each            cell which as an initial step requires production of mRNA            encoding the proteins to be synthesized.        -   Detection of incorporation of thymidine. The proliferative            capacity of T cells in response to stimulation by MHC            multimer can be determined by a radioactive assay based on            incorporation of [3H]thymidine ([3H]TdR) into newly            generated DNA followed by measurement of radioactive signal.        -   Detection of incorporation of BrdU. Cell proliferation can            also be detected by of incorporation of            bromo-2′-deoxyuridine (BrdU) followed by measurement of            incorporated BrdU using a labeled anti-BrdU antibody in an            ELISA based analysis.        -   Viability of cells may be measured by measurement ATP in a            cell culture.

Separation according to Size, Structure or other PhysicalCharacteristics

Properties of a sample can also be determined by measuring size and/orstructure of sample. Below different methods based on differentprinciples are listed.

Gel Electrophoresis

Gel electrophoresis is a technique used for the separation ofdeoxyribonucleic acid, ribonucleic acid, or protein molecules using anelectric current applied to a gel matrix. The term “gel” in thisinstance refers to the matrix used to contain and separate the targetmolecules. In most cases the gel is a crosslinked polymer whosecomposition and porosity is chosen based on the specific weight andcomposition of the target to be analyzed. When separating proteins orsmall nucleic acids (DNA, RNA, or oligonucleotides) the gel is usuallycomposed of different concentrations of acrylamide and a cross-linker,producing different sized mesh networks of polyacrylamide. Whenseparating larger nucleic acids (greater than a few hundred bases), thepreferred matrix is purified agarose. In both cases, the gel forms asolid, yet porous matrix. Acrylamide, in contrast to polyacrylamide, isa neurotoxin and must be handled using appropriate safety precautions toavoid poisoning.

“Electrophoresis” refers to the electromotive force (EMF) that is usedto move the molecules through the gel matrix. By placing the moleculesin wells in the gel and applying an electric current, the molecules willmove through the matrix at different rates, usually determined by mass,toward the positive anode if negatively charged or toward the negativecathode if positively charged.

After the electrophoresis is complete, the molecules in the gel can bestained to make them visible. Ethidium bromide, silver, or coomassieblue dye may be used for this process. Other methods may also be used tovisualize the separation of the mixture's components on the gel. If theanalyte molecules fluoresce under ultraviolet light, a photograph can betaken of the gel under ultraviolet lighting conditions. If the moleculesto be separated contain radioactivity added for visibility, anautoradiogram can be recorded of the gel.

If several mixtures have initially been injected next to each other,they will run parallel in individual lanes. Depending on the number ofdifferent molecules, each lane shows separation of the components fromthe original mixture as one or more distinct bands, one band percomponent. Incomplete separation of the components can lead tooverlapping bands, or to indistinguishable smears representing multipleunresolved components.

Bands in different lanes that end up at the same distance from the topcontain molecules that passed through the gel with the same speed, whichusually means they are approximately the same size. There are molecularweight size markers available that contain a mixture of molecules ofknown sizes. If such a marker was run on one lane in the gel parallel tothe unknown samples, the bands observed can be compared to those of theunknown in order to determine their size. The distance a band travels isapproximately inversely proportional to the logarithm of the size of themolecule.

Gel electrophoresis is usually performed for analytical purposes, butmay be used as a preparative technique prior to use of other methodssuch as mass spectrometry, RFLP, PCR, cloning, DNA sequencing, orSouthern blotting for further characterization.

Depending on the type of material to be separated different techniquesmay be used some of these are listed below:

-   -   Polypeptide length can be determined by SDS PAGE. The solution        of proteins to be analyzed is first mixed with SDS, an anionic        detergent which denatures secondary and non-disulfide-linked        tertiary structures, and applies a negative charge to each        protein in proportion to its mass. Without SDS, different        proteins with similar molecular weights would migrate        differently due to differences in mass charge ratio, as each        protein has an isoelectric point and molecular weight particular        to its primary structure. This is known as Native PAGE. Adding        SDS solves this problem, as it binds to and unfolds the protein,        giving a near uniform negative charge along the length of the        polypeptide.    -   SDS binds in a ratio of approximately 1.4 g SDS per 1.0 g        protein (although binding ratios can vary from 1.1-2.2 g SDS/g        protein), giving an approximately uniform mass:charge ratio for        most proteins, so that the distance of migration through the gel        can be assumed to be directly related to only the size of the        protein. A tracking dye may be added to the protein solution to        allow the experimenter to track the progress of the protein        solution through the gel during the electrophoretic run.        -   Native Gel Electrophoresis is a technique used mainly in            protein electrophoresis where the proteins are not denatured            and therefore separated based on their charge-to-mass ratio.            The two main types of native gels used in protein            electrophoresis are polyacrylamide gels and agarose gels.    -   Polyacrylamide gel electrophoresis (PAGE) is used for separating        proteins ranging in size from 5 to 2,000 kDa due to the uniform        pore size provided by the polyacrylamide gel. Pore size is        controlled by controlling the concentrations of acrylamide and        bis-acrylamide powder used in creating a gel. Care must be used        when creating this type of gel, as acrylamide is a potent        neurotoxin in its liquid and powdered form. The other type of        gel used is agarose gel. Agarose gels can also be used to        separate native protein. They do not have a uniform pore size,        but are optimal for electrophoresis of proteins that are larger        than 200 kDa.    -   Unlike SDS-PAGE type electrophoreses, Native gel electrophoresis        does not use a charged denaturing agent. The molecules being        separated (usually proteins) therefore differ in Molecular mass        and intrinsic charge and experience different electrophoretic        forces dependent on the ration of the two. Since the proteins        remain in the native state they may be visualised not only by        general protein staining reagents but also by specific        enzyme-linked staining.        -   QPNC-PAGE, or quantitative preparative native continuous            polyacrylamide gel electrophoresis, is a high-resolution            technique applied in biochemistry and bioinorganic chemistry            to separate proteins by isoelectric point. This variant of            gel electrophoresis is used by biologists to isolate active            or native metalloproteins in biological samples and to            resolve properly and improperly folded metal            cofactor-containing proteins in complex protein mixtures        -   Determination of size of DNA or RNA fragments, e.g. agarose            gel electrophoresis        -   Separation of proteins according to mass and isoelectric            point, e.g. 2D gel electrophoresis. Two-dimensional gel            electrophoresis, abbreviated as 2-DE or 2-D electrophoresis,            is a form of gel electrophoresis where mixtures of proteins            are separated by two properties in two dimensions on 2D            gels.    -   2-D electrophoresis begins with 1-D electrophoresis but then        separates the molecules by a second property in a direction 90        degree from the first. In 1-D electrophoresis, proteins (or        other molecules) are separated in one dimension, so that all the        proteins/molecules will lie along a lane but be separated from        each other by a property (e.g. isoelectric point). The result is        that the molecules are spread out across a 2-D gel. Because it        is unlikely that two molecules will be similar in two distinct        properties, molecules are more effectively separated in 2-D        electrophoresis than in 1-D electrophoresis.    -   The two dimensions that proteins are separated into using this        technique can be isoelectric point, protein complex mass in the        native state, and protein mass.

Chromatography

Chromatography is another method to separate structures according tosize, structure or other physical characteristics. In principle a sampledissolved in a “mobile phase” is passed through a stationary phase,which separates the analyte to be measured from other molecules in themixture and allows it to be isolated.

Below different types of chromatography is listed

-   -   Column chromatography        -   Ion exchange chromatography        -   Size exclusion chromatography        -   Liquid chromatography (LC, HPLC)        -   Gas chromatography    -   Planar chromatography        -   Paper chromatography        -   Thin layer chromatography

Separation Based on Chemical Properties

Samples can also be analysed be analyzing their chemical composition.The chemical composition of the whole sample med be determined,alternatively the chemical composition of fragments or individualstructures of the sample is identified. Often this type of analysis arepreceded by one or more other diagnostic or analytical methods e.g.separation according to size.

An example of this type of analysis is mass spectrometry. Massspectrometry is an analytical technique that identifies the chemicalcomposition of a compound or sample on the basis of the mass-to-chargeratio of charged particles. The method employs chemical fragmentation ofa sample into charged particles (ions) and measurements of twoproperties, charge and mass, of the resulting particles, the ratio ofwhich is deduced by passing the particles through electric and magneticfields in a mass spectrometer. The design of a mass spectrometer hasthree essential modules: an ion source, which transforms the moleculesin a sample into ionized fragments; a mass analyzer, which sorts theions by their masses by applying electric and magnetic fields; and adetector, which measures the value of some indicator quantity and thusprovides data for calculating the abundances each ion fragment present.The technique has both qualitative and quantitative uses, such asidentifying unknown compounds, determining the isotopic composition ofelements in a compound, determining the structure of a compound byobserving its fragmentation, quantifying the amount of a compound in asample using carefully designed methods (e.g., by comparison with knownquantities of heavy isotopes), studying the fundamentals of gas phaseion chemistry (the chemistry of ions and neutrals in vacuum), anddetermining other physical, chemical, or biological properties ofcompounds.

Measurement of Catalysis

Catalysis is the process in which the rate of a chemical reaction isincreased by means of a chemical substance known as a catalyst. Unlikeother reagents that participate in the chemical reaction, a catalyst isnot consumed. Thus, the catalyst may participate in multiple chemicaltransformations, although in practice catalysts are sometimes consumedin secondary processes. Examples of catalysis include but are notlimited to:

-   -   Measurement of enzymatic activity        -   Measurement of induction of enzymatic activity        -   Measurement of inhibition of enzymatic activity    -   Measurement of substrate for enzyme in a sample

Measurement of Growth

Growth is another parameter that can be measured in a sample. It can benatural growth in sample, the samples impact of growth on another sampleor analysis system and/or the growth in sample after the sample has beenadded stimulus, inhibitor or other substance influencing growth.

Examples of measurement of growth in sample include but are not limitedto:

-   -   Measurement of proliferation    -   Measurement of viability    -   Measurement of volume    -   Measurement of density

The above described chemical assays may be combined with one or moreother chemical assays in order to make the final diagnosis of the unit.The chemical assays may be applied to the sample of the unit or todifferent samples from the same unit.

Further Aspects Relates to Different Uses

Overall, the multimeric MHC's or compositions comprising such sets ofMHC's may find different uses. Thus, an aspect relates to the use of amultimeric major histocompatibility complex (MHC) or a compositionaccording to the invention for the detection of antigen-responsive cellsin a sample.

Another aspect relates to the use of a multimeric majorhistocompatibility complex (MHC) or a composition according to theinvention in the diagnosis of diseases or conditions, preferably cancerand/or infectious diseases.

A further aspect relates to the use of a multimeric majorhistocompatibility complex (MHC) or a composition according to theinvention in the development of immune-therapeutics.

Yet a further aspect relates to the use of a multimeric majorhistocompatibility complex (MHC) or a composition according to theinvention in the development of vaccines.

Another aspect relates to the use of a multimeric majorhistocompatibility complex (MHC) or a composition according to theinvention for the identification of epitopes.

In sum, the advantages of the present invention include, withoutlimitation, the possibility for detection of multiple (potentially, butexclusively, >1000) different antigen-responsive cells in a singlesample. The technology can be used, but is not restricted, for T-cellepitope mapping, immune-recognition discovery, diagnostics tests andmeasuring immune reactivity after vaccination or immune-relatedtherapies.

This level of complexity allow us to move from model antigens todetermination of epitope-specific immune reactivity covering fullorganisms, viral genomes, cancer genomes, all vaccine components etc. Itcan be modified in a personalized fashion dependent of the individualsMHC expression and it can be used to follow immune related diseases,such as diabetes, rheumatoid arthritis or similar.

Biological materials are for instance analyzed to monitor naturallyoccurring immune responses, such as those that can occur upon infectionor cancer. In addition, biological materials are analyzed for the effectof immunotherapeutics including vaccines on immune responses.Immunotherapeutics as used here is defined as active components inmedical interventions that aim to enhance, suppress or modify immuneresponses, including vaccines, non-specific immune stimulants,immunosuppressives, cell-based immunotherapeutics and combinationsthereof.

The invention can be used for, but is not restricted to, the developmentof diagnostic kits, where a fingerprint of immune response associated tothe given disease can be determined in any biological specimen. Suchdiagnostic kits can be used to determining exposure to bacterial orviral infections or autoimmune diseases, e.g., but not exclusivelyrelated to tuberculosis, influenza and diabetes. Similar approach can beused for immune-therapeutics where immune-responsiveness may serve as abiomarker for therapeutic response. Analyses with a barcode labelled MHCmultimer library allow for high-throughput assessment of large numbersof antigen responsive cells in a single sample.

Furthermore, barcode labelled MHC-multimers can be used in combinationwith single-cell sorting and TCR sequencing, where the specificity ofthe TCR can be determined by the co-attached barcode. This will enableus to identify TCR specificity for potentially 1000+ differentantigen-responsive T-cells in parallel from the same sample, and matchthe TCR sequence to the antigen specificity. The future potential ofthis technology relates to the ability to predict antigen responsivenessbased on the TCR sequence. This would be highly interesting as changesin TCR usage has been associated to immune therapy (11,12).

Further, there is a growing need for the identification of TCRsresponsible for target-cell recognition (e.g., but not exclusive, inrelation of cancer recognition). TCRs have been successfully used in thetreatment of cancer (13), and this line of clinical initiatives will befurther expanded in the future. The complexity of the barcode labeledMHC multimer libraries will allow for personalized selection of relevantTCRs in a given individual.

Due to the barcode-sequence readout, the barcode labeled MHC multimertechnology allow for the interaction of several different peptide-MHCcomplexes on a single cell surface, while still maintaining a usefulreadout. When one T-cell binds multiple different peptide-MHC complexesin the library, their relative contribution to T-cell binding can bedetermined by the number of reads of the given sequences. Based on thisfeature it is possible to determine the fine-specificity/consensussequences of a TCR. Each TCR can potential recognize large numbers ofdifferent peptide-MHC complexes, each with different affinity (14). Theimportance of such quantitative assessment has increased with clinicalused of TCRs and lack of knowledge may have fatal consequences asrecently exemplified in a clinical study where cross recognition of asequence related peptide resulted in fatal heart failure in two cases(15,16). Thus, this particular feature for quantitative assessment ofTCR binding of peptide-MHC molecules related to the present inventioncan provide an efficient solution for pre-clinical testing of TCRs aimedfor clinical use.

Also related to the above, this allows for determination of antigenresponsiveness to libraries of overlapping or to very similar peptides.Something that is not possible with present multiplexing technologies,like the combinatorial encoding principle. This allows for mapping ofimmune reactivity e.g. to mutation variant of viruses, such as, but notexclusive, HIV.

In broad embodiment, the present invention is the use of barcodelabelled MHC multimers for high-throughput assessment of large numbersof antigen responsive cells in a single sample, the coupling of antigenresponsiveness to functional and phenotypical characteristic, to TCRspecificity and to determine the quantitative binding of largepeptide-MHC libraries to a given TCR.

While the foregoing written description of the invention enables one ofordinary skill to make and use what is considered presently to be thebest mode thereof, those of ordinary skill will understand andappreciate the existence of variations, combinations, and equivalents ofthe specific embodiment, method, and examples herein. The inventionshould therefore not be limited by the above described embodiment,method, and examples, but by all embodiments and methods within thescope and spirit of the invention.

It is as aspect of the invention to provide the use of barcode labelledMHC multimers for multiplex detection of different T-cell specificitiesin a single sample, enabling simultaneous detection of potentially morethan 1000 different T-cell specificities where the specificity isrevealed through sequencing of the barcode label.

It is as aspect of the invention to provide the use of barcode labelledMHC multimers in combination with single-cell sorting and TCRsequencing, where the specificity of the TCR can be determined by theco-attached barcode. This will enable identification of TCRs specificfor a mixture of numerous (potentially, but not restricted to >1000)different peptide-MHC multimers, and match the TCR sequence to theantigen specificity.

It is as aspect of the invention to provide the use of barcode labelledMHC multimers for determining the affinity and binding motif of a givenTCR. The barcode labelling strategy will allow for attachment of severaldifferent (sequence related) peptide-MHC multimers to a givenT-cell—with the binding affinity determining the relative contributionby each peptide-MHC multimer. Thereby it is possible to map thefine-specificity/consensus recognition sequence of a given TCR by use ofoverlapping peptide libraries or e.g. alanine substitution libraries.

It is as aspect of the invention to provide the use of barcode labelledMHC multimers to map antigen responsiveness against sequencerelated/similar peptides in the same libraries, e.g. mutational changesin HIV infection. This has not been possible with previous MHC multimerbased techniques.

It is as aspect of the invention to provide the use of barcode-labelledMHC multimers to couple any functional feature of a specific T-cell orpool of specific T-cells to the antigen (peptide-MHC) recognition. E.g.determine which T-cell specificities in a large pool secrete cytokines,releases Calcium or other functional measurement after a certainstimuli.

Sample

A binding molecule is a molecule that specifically associates covalentlyor non-covalently with a structure belonging to or associated with anentity in a sample. In one embodiment said entity is selected from thegroup consisting of a cell, a cell population, a sample comprising oneor more cells, a molecule, a marker molecule, a surface of a biologicalcell, a cellular entity, and a cellular component (e.g. micelle),

In an embodiment the methods of the present invention includes providinga sample such as a biological sample.

The type of sample may vary. In an embodiment the sample is a biologicalsample. In an embodiment the sample is a blood sample, such as anperipheral blood sample, a blood derived sample, a tissue biopsy oranother body fluid, such as spinal fluid, or saliva.

The source of the sample may also vary. Thus, in a further embodimentsaid sample has been obtained from a mammal, such as a human, mouse,pigs, and/or horses.

In a preferred embodiment one cell may bind 1, 2-5, 6-20, 21-100,101-1000, 1001-50000, 50000-200000, or more than 200000 detectionmolecules.

Each individual cell to be combined with at least one detection moleculehas the capacity to bind a single or a number of detection molecules. Itfollows that one cell in one embodiment binds 1 detection moleculeaccording to the invention, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 detectionmolecules, such as binds 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10,10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-75, 75-100, 100-150,150-200, 200-250, 250-500, 500-750, 750-1000, 1000-2000, 2000-5000,5000-10.000, 10.000-25.000, 25.000-50.000, 50.000-100.000,100.000-250.000, 250.000-500.000, 500.000-1.000.000, or more than1.000.000 detection molecules.

In a preferred embodiment the binding molecule specifically associatescovalently or non-covalently with a structure belonging to or associatedwith a cell in a sample.

The sample in one embodiment is selected from the group consisting of asolid sample, a fluid sample, a semifluid sample, a liquid sample, asolubilised sample and a sample comprising dissociated cells of a solidsample.

The sample in one embodiment is selected from the group consisting of abiofilm, a biopsy, a surgical sample, a tissue sample, amicroarray-fixed sample, solid tissue section such as a fresh section, afrozen section and a FFPE section.

Cell samples capable of being analyzed by detection molecules and thenanalysed using flow cytometry include but is not limited to blood, CSF,lymph, cell lines (e.g. hybridomas, transfected cells), semen,suspension of bacteria, suspension of viral particles, suspension ofbeads or other particles or supra molecular structures, homogenizedtissues or any other fluidic sample from a given sample.

In one embodiment the sample is selected from the group consisting ofblood, whole blood, plasma, serum, Peripheral blood mononuclear cells(PBMC), human PBMN (HPBMC), buffy coat, synovial fluid, bone marrow,cerebrospinal fluid, saliva, lymph fluid, seminal fluid, urine, stool,exudate, transdermal exudates, pharyngeal exudates, nasal secretions,sputum, sweat, bronchoalveolar lavage, tracheal aspirations, fluid fromjoints, vitreous fluid, vaginal or urethral secretions or semen.

Herein, disaggregated cellular tissues such as, for example, hair, skin,synovial tissue, tissue biopsies and nail scrapings are also consideredas biological samples.

IN one embodiment the sample comprises one or more cells is selectedfrom the group consisting of immune cells, lymphocytes, monocytes,dendritic cells, T-cells, B-cells and NK cells.

In one embodiment said sample comprises one or more cells selected fromthe group consisting of CD4+ T cells, CD8+ T cells, αβ T cells,invariant γδ T cells, an antigen-specific T-cell, antigen-responsive Tcell and cells comprises T-cell receptors.

Many of the assays and methods described in the present invention areparticularly useful for assaying T-cells in blood samples. Blood samplesincludes but is not limited to whole blood samples or blood processed toremove erythrocytes and platelets (e.g., by Ficoll densitycentrifugation or other such methods known to one of skill in the art)and the remaining PBMC sample, which includes the T-cells of interest,as well as B-cells, macrophages and dendritic cells, is used directly.Also included are blood samples processed in other ways e.g. isolatingvarious subsets of blood cells by selecting or deselecting cells orentities in blood.

In one embodiment said cell is a cancer cell.

In one embodiment said sample is derived from an organ selected from thegroup consisting of lymph nodes, kidney, liver, skin, brain, heart,muscles, bone marrow, skin, skeleton, lungs, the respiratory tract,spleen, thymus, pancreas, exocrine glands, bladder, endocrine glands,reproduction organs including the phallopian tubes, eye, ear, vascularsystem, the gastroinstestinal tract including small intestines, colon,rectum, canalis analis and prostate gland; normal, diseased and/orcancerous.

In one embodiment the surface of sample cells is coated with proteasescapable of cleaving a peptide label, for example by addingantibody-protease conjugates where the antibody recognizes a particularcell surface structure.

In one embodiment the surface of sample cells is coated with DNAoligonucletides (“coating DNA”), for example by adding antibody-DNAconjugates where the antibody recognizes a particular cell surfacestructure.

Diseases

Detection molecules of the present invention can be used in immunemonitoring, diagnostics, prognostics, therapy and vaccines for manydifferent diseases, including but not limited to the diseases listed inthe following.

a) Infectious diseases caused by virus such as,

Adenovirus (subgroups A-F), BK-virus, CMV (Cytomegalo virus, HHV-5), EBV(Epstein Barr Virus, HHV-4), HBV (Hepatitis B Virus), HCV (Hepatitis Cvirus), HHV-6a and b (Human Herpes Virus-6a and b), HHV-7, HHV-8, HSV-1(Herpes simplex virus-1, HHV-1), HSV-2 (HHV-2), JC-virus, SV-40 (Simianvirus 40), VZV (Varicella-Zoster-Virus, HHV-3), Parvovirus B19,Haemophilus influenza, HIV-1 (Human immunodeficiency Virus-1), HTLV-1(Human T-lymphotrophic virus-1), HPV (Human Papillomavirus giving riseto clinical manifestations such as Hepatitis, AIDS, Measles, Pox,Chicken pox, Rubella, Herpes and others.

b) Infectious diseases caused by bacteria such as,

Gram positive bacteria, gram negative bacteria, intracellular bacterium,extracellular bacterium, Mycobacterium tuberculosis, Mycobacteriumbovis, Mycobacterium avium subsp. Paratuberculosis, Borreliaburgdorferi, Borrelia Garinii, Borrelia Afzelii, other spirochetes,Helicobacter pylori, Streptococcus pneumoniae, Listeria monocytogenes,Histoplasma capsulatum, Bartonella henselae, Bartonella quintana givingrise to clinical manifestations such as Tuberculosis, Pneumonia, Stomachulcers, Paratuberculosis and others.

c) Infectious diseases caused by fungus such as,

Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans,Pneumocystis carinii giving rise to clinical manifestations such asskin-, nail-, and mucosal infections, Meningitis, Sepsis and others.

d) Parasitic diseases caused by parasites such as,

Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae,Schistosoma mansoni, Schistosoma japonicum, Schistosoma haematobium,Trypanosoma cruzi, Trypanosoma rhodesiense, Trypanosoma gambiense,Leishmania donovani, and Leishmania tropica.

e) Allergic diseases caused by allergens such as,

Birch, Hazel, Elm, Ragweed, Wormwood, Grass, Mould, Dust Mite givingrise to clinical manifestations such as Asthma.

f) Transplantation-related diseases caused by

reactions to minor histocompatibility antigens such as HA-1, HA-8,USP9Y, SMCY, TPR-protein, HB-1Y and other antigens in relation to,Graft-versus-host-related disease, allo- or xenogene reactions i.e.graft-versus-host and host-versus-graft disease.

g) Cancerous diseases associated with antigens such as

Survivin, Survivin-2B, Livin/ML-IAP, Bcl-2, Mcl-1, Bcl-X(L), Mucin-1,NY-ESO-1, Telomerase, CEA, MART-1, HER-2/neu, bcr-abl, PSA, PSCA,Tyrosinase, p53, hTRT, Leukocyte Proteinase-3, hTRT, gp100, MAGEantigens, GASC, JMJD2C, JARD2 (JMJ), JHDM3a, WT-1,CA 9, Protein kinases,where the cancerous diseases include malignant melanoma, renalcarcinoma, breast cancer, lung cancer, cancer of the uterus, cervicalcancer, prostatic cancer, pancreatic cancer, brain cancer, head and neckcancer, leukemia, cutaneous lymphoma, hepatic carcinoma, colorectalcancer, bladder cancer.

h) Autoimmune and inflammatory diseases, associated with antigens suchas GAD64, Collagen, human cartilage glycoprotein 39, β-amyloid, Aβ42,APP, Presenilin 1, where the autoimmune and inflammatory diseasesinclude Diabetes type 1, Rheumatoid arthritis, Alzheimer, chronicinflammatory bowel disease, Crohn's disease, ulcerative colitis uterosa,Multiple Sclerosis, Psoriasis

EXAMPLES Example A

This example relates to

i) the stability of DNA oligonucleotides (DNA tag/label), used in oneembodiment of the invention, in blood preparations, and

ii) an embodiment of the invention, in which certain tagged Dextramers(detection molecules in which the binding molecule is a number ofpeptide-MHC complexes, and the label is a DNA oligonucleotide) areenriched for. Allowing identification of the Dextramers with bindingspecificity for certain (subpopulations of) cells in the cell sampletested.

In i) it is shown that DNA oligos are stable during handling in PBMC'sand in blood for a time that will allow staining, washing and isolationof T cells and subsequent amplification of DNA tags.

In ii) Show that a model system consisting of DNA-tagged Dextramers withMHC specificities for CMV, Flu and negative control peptide will locateto and can be captured/sorted with relevant T cell specificities and canbe identified by PCR amplification and/or sequencing.

Stability of Single-Stranded and Double-Stranded Oligonucleotides inBlood Preparations

DNA tag oligo design. 69-nucleotide long, biotinylated TestOligo wasprepared, consisting of

5′primer region (22 nt italic type, left)—

random region (6×N-nt)—barcode region (21 nt underlined)—

3′primer region (20 nt italic type, right):

Forward-01 primer GAGATACGTTGACCTCGTTG Reverse-01 primerATGCAACCAAGAGCTTAAGT TestOligo-01bGAGATACGTTGACCTCGTTGAANNNNNNTCTATCCATTCCATCCAGCTC ACTTAAGCTCTTGGTTGCATTestOligo-02 bhGAGATACGTTGACCTCGTTGAANNNNNNTCTATCCATTCCATCCAGCTCACTTAAGCTCTTGGTTGCAT TestOligo-03bhGAGATACGTTGACCTCGTTGAANNNNNNTCTATCCATTCCATCCAGCTCACTTAAGCTCTTGGTTGCATh TestOligo-04bhGAGATACGTTGACCTCGTTGAANNNNNNTCTTGAACTATGAATCGTCTCACTTAAGCTCTTGGTTGCATh TestOligo-05bhGAGATACGTTGACCTCGTTGAANNNNNNTCTATAGGTGTCTACTACCTCACTTAAGCTCTTGGTTGCATh TestOligo-06bhGAGATACGTTGACCTCGTTGAANNNNNNTCTTTATTGGAGAGCACGCTCACTTAAGCTCTTGGTTGCATh ′b′ = Biotin-TEG 5′ modification ′h′ = HEG(terminal modifications)

Q-PCR probes for quantifying the amount of TestOligos 1-6:

+=locked nucleic acid (LNA) modified RNA nucleotide

LNA-3

8=FAM; 7=BHQ-1-plusTCT[+A][+T][+C]A[+T][+T]CC[+A][+T][+C]CAGC

LNA-4

8=FAM; 7=BHQ-1-plusTCT[+T][+G][+A]AC[+T][+A]TG[+A][+A][+T]CGTC

LNA-5

9=HEX; 7=BHQ-1-plusTCT[+A][+T][+A]GG[+T][+G]TC[+T][+A][+C]TACC

LNA-6

2=Cy5; 1=BHQ-2-plusTCT[+T][+T][+A]TT[+G][+G]AG[+A][+G][+C]ACGC

The stability of oligo-tags by Q-PCR was analyzed under conditionsrelevant for T cell isolation:

The testOligos 1-6 were incubated in anticoagulated EDTA blood, andfollowing incubation the amount of each of the testOligos was determinedusing Q-PCR using the abovementioned primers and probes. The oligo tagswere quantified by QPCR with SYBR® Green JumpStart™ Taq ReadyMix™according to manufacturer's protocol in combination with any capillaryQPCR instruments (e.g. Roche LightCycler or Agilent Mx3005P).

Because of the different termini of the testOligos 1-6, this also was atest of the stability of non-modified DNA oligo tag vs HEG modified 5′and HEG modified 5′ and 3′ (TestOligo-01, -02 and -03 respectively).

The results are shown in FIG. 8 . It is concluded that the stability ofthe testOligos is appropriately high for all variants tested, to performthe invention.

Generation and Screening of a 3 Member DNA Tagged MHC Dextramer Libraryfor Screening of Antigen Specific T Cells in a Lymphoid Cell Sample

This experiment involves the generation of 3 DNA-tagged Dextramers, eachwith a unique specificity, as follows:

Dextramer 1: Flu (HLA-A*0201/GILGFVFTL/MP/Influenza)

Dextramer 2: CMV (HLA-A*0201/NLVPMVATV/pp65/CMV)

Dextramer 3: Negative (HLA-A*0201/ALIAPVHAV/Neg.Control).

Each of these Dextramers thus have a unique pMHC specificity (i.e. thethree Dextramers have different binding molecules), and each Dextramercarries a unique label (DNA oligonucleotide) specific for that one pMHCspecificity.

The library of DNA-tagged Dextramers are screened in a preparation oflymphoid cells such as anticoagulated EDTA blood or preparations ofperipheral blood mononucleated cells (PBMC's). Those Dextramers thatbind to cells of the cell sample will be relatively more enriched thanthose that do not bind.

Finally, the MHC/antigen specificity of the enriched Dextramers isrevealed by identification of their DNA tags by Q-PCR with DNAtag-specific probes or by sequencing of the DNA tags.

1. Production of 3 different DNA tagged Dextramers withHLA-A*0201-peptide (pMHC) complexes.

pMHC complexes are generated and attached to dextran, along with uniqueDNA tags identifying each of the individual pMHC complexes, as follows.

Generation of DNA tagged Dextramers with Flu(HLA-A*0201/GILGFVFTL/MP/Influenza), CMV (HLA-A*0201/NLVPMVATV/pp65/CMV)and Negative (HLA-A*0201/ALIAPVHAV/Neg.Control).

Dextramer stock is 160 nanomolar (nM), TestOligo stock is diluted to 500nM. Mix 10 micro liter (uL) 160 nM dextramer stock with 10 uL 500 nMTestOligo stock. Incubate 10 min at r.t. Mix with 1.5 ug pMHC complex ofdesired specificity. Adjust volume to 50 uL with a neutral pH buffersuch as PBS or Tris pH 7.4, and store at 4 degrees Celsius. This willproduce a DNA tagged Dextramer with approximately 3 oligo tags and 12pMHC complexes, respectively, per Dextramer.

-   -   a. Dex-Oligo-03=Dextramer with TestOligo-03 and        HLA-A*0201/NLVPMVATV/pp65/CMV.    -   b. Dex-Oligo-04=Dextramer with TestOligo-04 and        HLA-A*0201/GILGFVFTL/MP/Influenza.    -   c. Dex-Oligo-05=Dextramer with TestOligo-05 and        HLA-A*0201/ALIAPVHAV/Neg.Control.

2. Preparation of cell sample for screening for antigen-specific Tcells.

-   a. Appropriate cell samples for identification of antigen specific T    cells are preparations of lymphoid cells such as preparations of    peripheral blood mononucleated cells (PBMC's) or anticoagulated    blood. Such preparations of cell samples are prepared by standard    techniques known by a person having ordinary skill in the art.-   b. Transfer in the range of 1E7 lymphoid cells (from PBMC or EDTA    anticoagulated blood) to a 12×75 mm polystyrene test tube.-   c. Add 2 ml PBS containing 5% fetal calf serum, pH 7.4. Centrifuge    at 300×g for 5 min. Remove supernatant and resuspend cells in a    total volume of 2.5 ml PBS containing 5% fetal calf serum, pH 7.4.

3. Preparation and modification of library of DNA tagged Dextramers withthree MHC/peptide specificities (from 1).

-   a. Mix 5 ul 10 uM biotin with 10 ul each of Dex-Oligo-03,    Dex-Oligo-04 and Dex-Oligo-05.

4. Mixing of preparations of lymphoid cells with a library of DNA taggedMHC Dextramers.

-   a. Mix 1E7 lymphoid cells in 2.5 mL (from 2b) with 30 uL library of    DNA tagged Dextramers (from 3a).-   b. Incubate 30 min at r.t.-   c. Centrifuge at 300×g for 5 min. and remove the supernatant.-   d. Resuspend pellet in 2.5 ml PBS containing 5% fetal calf serum, pH    7.4. Centrifuge at 300×g for 5 min. and remove the supernatant.-   e. Resuspend pellet in 2.5 ml PBS containing 5% fetal calf serum, pH    7.4

5. Capture of all CD8+ antigen specific T cells by magnet assisted cellsorting, performed according to Miltenyi Biotec catalog nr 130-090.878,Whole Blood CD8 MicroBead protocol.

-   a. Add 100 uL Whole Blood CD8 MicroBeads (Miltenyi Biotec catalog nr    130-090.878) to resuspended lymphoid cells from 4e. Mix and allow    capture of CD8+ T cells for 15 min at r.t.-   b. Place Whole Blood Column in the magnetic field of a suitable MACS    Separator. For details see the Whole Blood Column Kit data sheet.-   c. Prepare column by rinsing with 3 mL separation buffer (autoMACS    Running Buffer or PBS containing 5% fetal calf serum, pH 7.4).-   d. Apply magnetically labeled cell suspension (4e) onto the prepared    Whole Blood Column. Collect flow-through containing unlabeled cells.-   e. Wash Whole Blood Column with 3×3 mL separation buffer (autoMACS    Running Buffer or PBS containing 5% fetal calf serum, pH 7.4).-   f. Remove Whole Blood Column from the separator and place it on a    new collection tube.-   g. Capture CD8+ T cells by pipetting 5 mL Whole Blood Column Elution    Buffer or PBS containing 5% fetal calf serum, pH 7.4 onto the Whole    Blood Column. Immediately flush out the magnetically labeled cells    by firmly pushing the plunger into the column.-   h. Centrifuge at 300×g for 5 min. and remove the supernatant.    Resuspend the collected CD8+ cells in 50 uL and store at minus 20    degrees Celsius for subsequent analysis.

6. Identification of Dextramers that bound significantly to antigenspecific T cells of the lymphoid cell sample.

-   a. Quantifying ratios of DNA oligo tags in input (3a) vs captured    fraction (5 h) by sequencing or alternatively quantifying by QPCR    using the DNA tag specific probes LNA-3, LNA-4 and LNA-5 will reveal    the relative abundance of antigen specific T cells in the lymphoid    cell sample.    -   i. Quantifying ratios of DNA oligo tags in input (3a) vs        captured fraction (5 h) by QPCR using the DNA tag specific        probes LNA-3, LNA-4 and LNA-5.        -   1. Make 25 uL QPCR reactions of            -   a. input of library of DNA tagged Dextramers (3a)            -   b. output of library of DNA tagged Dextramers (5 h)            -   c. Standard curves of 10 to 1E8 TestOligo-03,                TestOligo-04 and TestOligo-05 respectively.        -   2. Mix 12.5 uL JumpStart Taq ReadyMix (Sigma-Aldrich #D7440)            with 0.125 uL 100 uM primer each of Forward-01 and            Reverse-01, 0,625 ul 10 uM of either probe LNA-3, LNA-4 or            LNA-5, 0.025 ul Reference dye (Sigma-Aldrich #R4526) and            12.5 uL of either input of library of DNA tagged Dextramers            (3a), output of library of DNA tagged Dextramers (5 h) or            Standard curves of 10 to 1E8 TestOligo-03, TestOligo-04 and            TestOligo-05 respectively.        -   3. Run two step QPCR thermal profile Cycle 1=5 min at 95            degrees Celsius, Cycle 2-40=30 sec at 95 degrees Celsius and            1 min at 60 degrees Celsius.        -   4. Estimate the relative abundance of T cells with antigen            specificity against one of the three MHC Dextramers by            plotting the QPC cycle time (Ct) values of the input of            library of DNA tagged Dextramers (3a), the output of library            of DNA tagged Dextramers (5 h) in a plot of Ct values of the            QPCR standard curve of TestOligo-03, TestOligo-04 and            TestOligo-05 respectively.    -   ii. Quantifying ratios of DNA oligo tags in input (3a) vs        captured fraction (5 h) by ultra-deep sequencing.        -   1. Make 25 uL PCR reactions of            -   a. input of library of DNA tagged Dextramers (3a)            -   b. output of library of DNA tagged Dextramers (5 h)        -   2. Mix PCR reaction using any standard PCR master mix with            1.25 uL 10 uM primer each of Forward-01 and Reverse-01, and            12.5 uL of either input of library of DNA tagged Dextramers            (3a) or output of library of DNA tagged Dextramers (5 h).            Top up to 25 uL with pure water. For example use 2x PCR            Master Mix from Promega containing Taq DNA polymerase,            dNTPs, MgCl2 and reaction buffers.        -   3. Ultra Deep Sequencing of the above PCR product can be            provided by a number of commercial suppliers such as for            example Eurofins Genomics, GATC Biotech or Beckman Coulter            Genomics using well established Next Generation Sequencing            technologies such as Roche 454, Ion Torrent, the Illumina            technology or any other high throughput sequencing technique            for PCR amplicon sequencing.        -   4. PCR amplicon analysis of the relative abundances of the            input of library of DNA tagged Dextramers (3a), the output            of library of DNA tagged Dextramers (5 h) will reveal the            relative abundance of T cells with antigen specificity            against one of the three MHC Dextramers.

7. Predicted results and comments

-   a. It is expected that the relative abundance and ratios of DNA    oligo tags in input of a library of DNA tagged Dextramers (3a) as    estimated by QPCR or sequencing is primarily affected by three    parameters namely i) the ratio in which the DNA oligo tags were    supplied during the generation of the DNA tagged Dextramers    (1.a.i.1), ii) how the library input was mixed (3a) and iii) how    efficiently the individual DNA oligo tags are amplified in the PCR    reactions.    -   i. In an example, the relative ratios of DNA oligo tags in input        of a library of DNA tagged Dextramers as generated in 3a and as        measured by QPCR or sequencing would be between 1 to 10 fold of        each other.-   b. It is expected that the relative abundance and ratios of DNA    oligo tags in the output of library of DNA tagged Dextramers (5 h)    as estimated by QPCR or sequencing, in addition to the three    parameters mentioned in 7a, is primarily affected by three    additional parameters namely i) the number of antigen specific T    cells with specificity for one of the three MHC-peptide    combinations ii) the affinity of the T cell receptor of the given T    cell for the given MHC-peptide complex and finally iii) the    efficiency of separating antigen-specific T cells and their    associated DNA tagged MHC Dextramers from unbound DNA tagged MHC    Dextramers by washing and cell capture.    -   i. In an example, the relative ratios of DNA oligo tags in        output of a library of DNA tagged Dextramers as generated in 5 h        and as measured by QPCR or sequencing would be more than 10 fold        in favor of those DNA oligo tags coupled to an MHC Dextramer        with an MHC-peptide complex for which antigen-specific T cells        are present in the lymphoid cell sample.        -   1. In a lymphoid cell sample from an influenza positive and            CMV positive HLA-A0201 donor with antigen-specific T cells            against HLA-A*0201/NLVPMVATV/pp65/CMV and            HLA-A*0201/GILGFVFTL/MP/Influenza and no antigen-specific T            cells against HLA-A*0201/ALIAPVHAV/Neg.Control it is            expected that the relative ratios of TestOligo-03            (Dex-Oligo-03=Dextramer with TestOligo-03 and            HLA-A*0201/NLVPMVATV/pp65/CMV), TestOligo-04            (Dex-Oligo-04=Dextramer with TestOligo-04 and            HLA-A*0201/GILGFVFTL/MP/Influenza) and TestOligo-05            (Dex-Oligo-05=Dextramer with TestOligo-05 and            HLA-A*0201/ALIAPVHAV/Neg.Control) will be more than 10 fold            in the favor of TestOligo-03 and TestOligo-04 over            TestOligo-05. That is TestOligo-03 and TestOligo-04 is            expected to be more than 10 fold more abundant or frequent            than TestOligo-05 as measured by sequencing or QPCR of the            output of library of DNA tagged Dextramers (5 h) if they            were supplied in equal amounts in the input of library of            DNA tagged Dextramers (3a).

Example B

FIG. 7 shows results that act as proof-of-principle for the claimedinvention. FIG. 7A, Flow cytometry data of peripheral blood mononuclearcells (PBMCs) from healthy donors.

Materials and Methods

PBMCs were stained with CMV specific peptide-MHC multimers coupled to aspecific nucleotide-barcode. In addition to CMV peptide-MHC reagents thecells were stained in the presence of negative control reagents i.e.HIV-peptide MHC multimers coupled to another specific barcode label andthe additional negative control peptide-MHC reagents (p*) not holding abarcode—all multimers were additionally labeled with a PE-fluorescencelabel. The amounts of MHC multimers used for staining of PBMCs wereequivalent to the required amount for staining of 1000 differentpeptide-MHC specificities i.e. 1× oligo-labeled CMV specific MHCmultimers, 1× oligo-labeled HIV specific MHC multimers and 998×non-labeled p*MHC multimers, so as to give an impression whetherbackground staining will interfere with the true positive signal.Prolonged washing steps were included (either 0 min (A), 30 min (B) or60 min (C)) after removing the MHC multimers, and data from allexperiments are shown. The PE-MHC-multimer positive cells were sorted byfluorescence activated cell sorting (FACS) FIG. 7B, Cross threshold (Ct)values from multiplex qPCR of the sorted PE-MHC-multimer positive cells.QPCR was used to assess the feasibility of detecting certain cellspecificity through barcode-labeled peptide-MHC-multimers. Reagentsassociated with a positive control (CMV) barcode and a negative control(HIV) barcode were present during staining, but negative control (HIV)barcode-peptide-MHC multimers should be washed out.

Examples of nucleic acid sequences are:

DNA-barcode oligo for CMV MHC multimer attachment:

5GAGATACGTTGACCTCGTTGAANNNNNNTCTATCCATTCCATCCAGCT CACTTAAGCTCTTGGTTGCAT

DNA-barcode oligo for HIV MHC multimer attachment:

5GAGATACGTTGACCTCGTTGAANNNNNNTCTATAGGTGTCTACTACCTC ACTTAAGCTCTTGGTTGCAT5 = Biotin-TEG

Results

Results shows Ct value only detectable to the CMV peptide-MHC multimerassociated barcode, whereas the HIV-peptide MHC multimer associatedbarcode was not detected

Conclusion

This experiment is a representative example of several similarexperiment performed with other antigen specificities. Overall thesedata show that it is feasible to

-   -   1) stain with at least 1000 different MHC-multimers in a single        sample while still maintain a specific signal,    -   2) attach a DNA-barcode to an MHC multimer,    -   3) amplify the DNA-barcode after cellular selection steps,    -   4) read the barcode with QPCR, using barcode specific probes,    -   5) obtain a specific signal corresponding to the antigen        specific T cell population present in the sample, while        non-specific MHC multimer barcodes are non-detectable.

Together these (and similar data available) provide proof of feasibilityfor the steps described in FIGS. 3, 4 and 5 .

Examples 1-12, 20-21, 60-61, 79-82 and 120-124

In the following examples, the experimental methods comprise one or moreof the following steps

-   -   1. Sample preparation.        -   a. Acquiring sample        -   b. Modifying sample    -   2. Linker preparation.    -   3. Binding molecules preparation        -   a. Synthesis        -   b. Modification        -   c. Purification    -   4. Label preparation        -   a. Synthesis        -   b. Modification        -   c. Purification    -   5. Detection molecules preparation        -   a. Synthesis        -   b. Modification        -   c. Purification    -   6. Incubation of sample and detection molecules        -   a. Amount of sample        -   b. Amount of detection molecule        -   c. Conditions    -   7. Enrichment of detection molecules with desired        characteristics        -   a. Apply        -   b. Wash        -   c. Separate    -   8. Identification of enriched detection molecule        -   a. Apply        -   b. Analysis

Example 1

In this example the binding molecules are pMHC complexes, the linker isa dextran-streptavidin-fluorochrome conjugate, and the label is a DNAoligonucleotide.

The sample is a HPBMC from humans, the isolating and/or detecting isdone by flow cytometry (FACS), and the determining of the identity ofthe label is done by quantitative PCR (QPCR).

Example 1 Explained

This is an example where the Sample (1) was blood from one CMV positiveand HIV negative donor which was modified (1b) to generate Peripheralblood mononuclear cells (PBMCs).

The Linker (2) was a dextran conjugate with streptavidin andfluorochrome (Dextramer backbone from Immudex).

The binding molecules (3) were peptide-MHC (pMHC) complexes displayingeither CMV (positive antigen) or HIV (negative antigen) derivedpeptide-antigens. The binding molecules were modified (3b) bybiotinylation to provide a biotin capture-tag for the Linker. Thebinding molecules were purified (2c) by HPLC and quality controlled interms of the formation of functional pMHC multimers for staining of acontrol T-cell population.

The Labels (4) were oligonucleotides applied as DNA-barcodes. Theoligonucleotides were synthetized (4a) by DNA Technology A/S (Denmark)and were synthetically modified (4b) with a terminal biotin capture-tag.The labels were combined oligonucleotide labels arising by annealing anA oligonucleotide (modified with biotin) to a partially complimentary Boligonucleotide label followed by enzymatic DNA polymerase extension ofOligo A and Oligo B to create a fully double stranded label. Thedetection molecule (5) was synthetized (5a) by attaching bindingmolecules in the form of biotinylated pMHC and labels in the form ofbiotin-modified double stranded oligonucleotides onto astreptavidin-modified dextran linker. The detection molecule furthercontained a modification (5b) in the form of a fluorochrome. Twodifferent detection molecules were generated wherein the two individualdetection molecules containing different pMHC were encoded bycorresponding individual oligonucleotide labels.

An amount of sample, PBMC's (1b) was incubated with an amount of mixeddetection molecules (5) under conditions (6c) that allowed binding ofdetection molecules to T cells in the sample.

The cell-bound detection molecules were separated from the non-cellbound detection molecules (7) by first a few rounds of washing thePBMC's through centrifugation sedimentation of cells and resuspension inwash buffer followed by Fluorescence Activated Cell Sorting (FACS) offluorochrome labeled cells. T cells that can efficiently bind detectionmolecules will fluoresce because of the fluorochrome comprised withinthe detection molecules; T cells that cannot bind detection moleculeswill not fluoresce. FACS-sorting leads to enrichment of fluorescentcells, and hence, enrichment of the detection molecules along with theassociated labels that bind T cells of the PBMC sample.

FACS isolated cells were subjected to quantitative PCR analysis of theoligonucleotide label associated with the detection molecules bound tothe isolated cells to reveal the identity of the detection moleculesthat bound to the T cells present in the sample. This example thusrevealed the presence of T cells in the blood expressing a T cellreceptor that binds to peptide-MHC molecules represented within thelibrary of Detection Molecules. It also revealed the feasibility ofenriching for Detection Molecules based on the presence of T cellsspecific for the positive peptide-antigen (CMV) over the negativepeptide-antigen (HIV) antigens.

Example 1 in Detail

-   -   1. Sample preparation. The cell sample used in this example was        obtained by preparing PBMC's from blood drawn from a donor that        was CMV positive as well as HIV negative as determined by        conventional MHC-multimer staining.        -   a. Acquiring sample: Blood was obtained from the Danish            Blood Bank, in the form of buffy coats (BC). A peripheral            blood mononuclear cell preparation obtained following            standard donor blood preparations.        -   b. Modifying sample: Peripheral blood mononuclear cells            (PBMCs) were isolated from whole blood by density gradient            centrifugation. The density gradient medium, Lymphoprep            (Axis-Shield), which consists of carbohydrate polymers and a            dense iodine compound, facilitate separation of the            individual constituents of blood. Blood samples were diluted            1:1 in RPMI (RPMI 1640, GlutaMAX, 25 mM Hepes; gibco-Life            technologies) and carefully layered onto the Lymphoprep.            After centrifugation, 30 min, 490 g, PBMCs together with            platelets were harvested from the middle layer of cells. The            isolated cells, the buffy coat (BC), was washed twice in            RPMI and cryopreserved at −150° C. in fetal calf serum (FCS;            gibco-Life technologies) containing 10% dimethyl sulfoxide            (DMSO; Sigma-Aldrich). BC's used in this example are listed            in table 1 along with peptide-antigen specific T cells            identified within these samples by conventional MHC multimer            staining.    -   2. Linker preparation: The linker used in this example was a        dextran molecule, to which was attached streptavidin and        fluorochromes. The streptavidin served as attachment sites for        biotinylated oligonucleotides (Label) and biotinylated        peptide-MHC complexes (Binding Molecules). The fluorochrome        allowed separation of cells bound to detection molecules from        cells not bound to detection molecules.        -   i. In this example linkers were linear and branched dextran            molecules with covalently attached streptavidin (5-10 per            linker) and fluorochromes (2-20 per linker) in the form of            PE. Linkers were essentially Dextramer backbone as described            by Immudex.    -   3. Binding molecules preparation: The binding molecules used in        this example were two different class I MHC-peptide complexes.        MHC heavy chains (HLA-A0201 and HLA-B0702) and B2M were        expressed in E.coli as previously described (Hadrup et al. 2009)        and each MHC-I complex generated with two peptide antigens. The        individual specificities (allele and peptide combination) were        generated in the following way:        -   a. Synthesis: Binding molecules in this example were            specific pMHC monomers that were produced from UV-exchange            of selected HLA-I monomers carrying a photolabile 9-residue            peptide-ligand (p*). When exposed to UV-light (366 nm) the            photolabile ligand will be cleaved and leave the binding            groove empty. Due to the instability of empty MHC-I            molecules, the complexes will quickly degrade if they are            not rescued by replacement with another peptide that match            that HLA-type. In this way specific pMHC monomers were            produced by mixing excess of desired HLA ligands with p*MHC            monomers. p*MHC monomers were refolded, biotinylated and            purified as previously described (Hadrup et al. 2009).            -   i. HIV derived peptide ILKEPVHGV from antigen HIV                polymerase and CMV derived peptide TPRVTGGGAM from                antigen pp65 TPR (Pepscan Presto, NL) were diluted in                phosphate buffered saline (DPBS; Lonza) and mixed to                final concentrations 100 μg/ml:200 μM (HLA-A0201:                ILKEPVHGV and HLA-B0702:TPRVTGGGAM). The mixtures were                exposed to 366 nm UV light (UV cabinet; CAMAG) for one                hour and optionally stored for up to 24 h at 4° C.        -   b. Modification: No further modifications        -   c. Purification: Before applying the peptide exchanged HLA            monomer Binding Molecules for preparation of detection            molecules these were centrifuged 5 min, 3300 g, to sediment            any aggregated MHC molecules.    -   4. Label preparation: In this example, two different double        stranded oligonucleotides, DNA barcodes, of the same length but        partially different sequence, were generated. Each of the DNA        barcodes would become attached to a specific pMHC, and thus        functioned as a Label for this specific Binding Molecule. The        oligonucleotides were biotinylated, allowing easy attachment to        the dextran-streptavidin conjugate linker.        -   a. Synthesis: The Labels were generated from partially            complementary oligonucleotides which were purchased from DNA            Technology (Denmark) and delivered as lyophilized powder.            Stock dilutions of 100 μM oligonucleotides were made in            nuclease free water and stored at −20° C.            -   i. The Label system used in this example was named 2OS                and was developed to increase the complexity of a                limited number of oligonucleotide sequences. This was                enabled by applying a combinatorial strategy where two                partially complementary oligonucleotides (an A                oligonucleotide with a 5′ biotin tag and a B                oligonucleotide) where annealed and then elongated to                produce new unique oligonucleotide-sequences (AxBy)                which were applied as a DNA-barcodes (Labels) (see FIG.                9 for design of the DNA-barcodes).By combining 2 unique                oligonucleotide-sequences (A label precursor) that were                partly complementary to 2 other unique oligonucleotide                sequences (B label precursor) a combinatorial library of                4 different (AxBy) Labels were produced. Only 2 of these                Labels were used in this example (A1B1 and A2B2). Refer                to table 2 for an overview of the different 2OS A and B                nucleotide sequences. Briefly:            -   Partly complementary A and B oligonucleotides were                annealed to produce two combined A+B DNA barcodes (A1+B1                to produce A1B1 and A2+B2 to produce A2B2). The                respective A and B oligonucleotides were mixed as stated                in table 3, heated to 65° C. for 2 min and cooled slowly                to <35° C. in 15-30 min. The annealed A and B                oligonucleotides were then elongated as stated in                table 3. Elongation reagents were mixed <15 min before                use. After mixing, the reactions were incubated 5 min,                RT, to allow elongation of the annealed                oligonucleotides. The reagents used for annealing (left)                and elongation (right) of partly complementary                oligonucleotides is described in table 3. Reagents                marked in italic were from the Sequenase Version 2.0 DNA                Sequencing Kit (Affymetrix #70770).        -   b. Modification: All labels were diluted to working            concentrations (2.17 uM) in nuclease free water with 0.1%            Tween and stored at −20° C.        -   c. Purification: Labels were not purified further.    -   5. Detection Molecules preparation: The Binding Molecules        (pMHCs) and Labels (DNA-barcodes) were attached to the Linker        (dextran-streptavidin-PE conjugate), to form Detection        Molecules, in such a way that a given pMHC was always attached        to a given DNA-barcode.        -   a. Synthesis: For preparation of Detection Molecules the 2OS            DNA-barcodes were attached to the dextramer prior to            addition of pMHC. 1 ul dextramer was used for every 3 ul of            prepared Detection Molecule. Briefly, for generation of            Detection Molecules:            -   i. The Label:linker conjugate were generated by addition                of label in two fold excess over linker (label:linker,                2:1) i.e. 1×0.16 uM linker (dex), were mixed with                0.15×2.17 uM label (2OS DNA-barcode) and incubated at                least 30 min, 4° C.            -   ii. Binding molecules, in the form of biotinylated                UV-exchanged peptide-MHC monomer, were added to the                label:linker conjugate to reach a concentration of 44                ug/ml of the given pMHC in 3 ul, and incubated 30 min,                RT.            -   The final volume was reached by addition of 0.02% NaN₂                in PBS together with D-biotin (Avidity Bio200) in a                final concentration of 12.6×10⁻⁶M. The detection                molecule preparation was incubated 30 min, 4° C., and                optionally stored for up to 4 weeks at 4° C. (overview                of the amounts used can be found in table 4).            -   Two sets of two detection molecules were generated. Each                set with the two specificities individually labeled. The                label was inverted between the two sets as described                below.                -   1. 1×CMV specific pMHCs (HLA-A0201: ILKEPVHGV)                    coupled to 2OS-A1B1, 1×HIV specific pMHCs                    (HLA-B0702:TPRVTGGGAM) coupled to 2OS-A2B2.                -   2. 1×CMV specific pMHCs (HLA-A0201: ILKEPVHGV)                    coupled to 2OS-A2B2, 1×HIV specific pMHCs                    (HLA-B0702:TPRVTGGGAM) coupled to 2OS-A1B1.        -   b. Modification: No further modifications were performed        -   c. Purification: The Detection Molecules were centrifuged 5            min, 3300 g, to sediment any aggregates before being added            to the cell sample,    -   6. Incubation of sample and Detection Molecules: The cell sample        and the Detection Molecules were mixed in one container to allow        Detection Molecules to bind to T cells.        -   a. Amount of sample: 1×10E6-2×10E6 cells in the form of            BC's, were used.        -   b. Amount of detection molecule: According to table 4. for            staining in 100 ul 1.32 ug/ml calculated in relation to each            binding molecule (specific peptide-MHC) on the Detection            Molecule was required per incubation, i.e. 3 ul of each            Detection Molecule.        -   c. Conditions: BCs were thawed in 10 ml, 37° C., RPMI with            10% fetal bovine serum (FBS), centrifuged 5 min, 490 g, and            washed twice in 10 ml RPMI with 10% FBS. All washing of            cells refer to centrifugation 5 min, 490 g, with subsequent            removal of supernatant. 1×10E6-2×10E6 cells were washed in            200 ul barcode-buffer (PBS/0.5% BSA/2 mM EDTA/100 μg/ml            herring DNA) and resuspended in this buffer to approximately            20 μl per sample. The barcode buffer is optimized to            increase the stability of the oligonucleotides associated            with the Detection molecules. The cells were incubated with            50 nM dasatinib, 30 min, 37° C., prior to incubation of            cells with Detection Molecules.            -   i. The Detection molecules were added in the required                amount. If necessary barcode-buffer was added to reach a                total volume of 100 ul and cells were incubated 15 min,                37° C. 20 ul antibody mixture containing                PerCP-conjugated anti-CD8 antibody and dump channel                FITC-conjugated antibodies (table 5) along with 0.1 μl                near-IR-viability dye (Invitrogen L10119) was added for                every 100 ul of PBMC:detection molecule sample. The                samples were incubated 30 min, 4° C., and cells were                washed twice in 200 ul barcode-buffer. Optionally cells                were fixed in 1% paraformaldehyde in DPBS overnight, 4°                C., and washed twice in barcode-buffer. Fixed cells were                stored for up to a week at 4° C.    -   7. Enrichment of detection molecules with desired        characteristics: The Detection Molecules were enriched by using        flow cytometry, more specifically,        Fluorescence-Activated-Cell-Sorting (FACS). Since all Detection        Molecules carried a PE-fluorescent label, the cells that bound        to Detection Molecules would fluoresce according to this        fluorochromes emission peak. By applying a FACS sorter this        feature could be used to separate cells that bound to detection        molecules from cells that did not bind Detection Molecules i.e.        to separate those cells that did fluoresce from those cells that        did not fluoresce. As a result the Detection Molecules that        bound to cells, and hence the associated Labels, were enriched        for.        -   a. Apply: Cells were sorted on a BD FACSAria, equipped with            three lasers (488 nm blue, 633 nm red and 405 nm violet).            The flow cytometry data analyses were performed using the BD            FACSDiva software version 6.1.2. The following gating            strategy was applied: Lymphocytes were identified in a            FSC/SSC plot. Additional gating on single cells            (FSC-A/FSC-H), live cells (near-IR-viability dye negative),            and CD4, CD14, CD16, CD19, CD40 negative (FITC)/CD8 positive            cells (PerCP) were used to define the CD8 T cell population            (table 5). The PE positive population, i.e. the cells that            bound to detection molecules, were defined within the PerCP            positive population        -   b. Wash: not applied        -   c. Separate: The PE positive cells were sorted by FACS, as            described in 7a, into tubes that contained 200 μl            barcode-buffer and that had been pre-saturated for 2 h-O.N.            in 2% BSA. The sorted cells were centrifuged 5 min, 5000 g,            to allow removal of excess buffer (<10 ul should reside in            the container). Cells were stored at −80° C.    -   8. Identification of enriched Detection Molecules: Since the        Detection Molecules were enriched based on specific binding of        the Binding Molecule (the pMHC) to cells, the identification of        the associated Labels (oligonucleotide barcodes) amongst the        sorted cells would also reveal the pMHCs that had bound to cells        in the PBMC sample. In this example the enriched Detection        Molecules were identified by quantitative PCR (QPCR) using        Label-specific fluorescent reporter probes.        -   a. Apply: Labels derived from sorted cells were analyzed by            QPCR with the Brilliant II QRT-PCR Low ROX Master Mix Kit            (Agilent technologies, #600837) according to table 6. PCR            was run on the thermal cycler: Mx3000P qPCR system (Agilent            Technologies). The thermal profile is listed in table 7 and            the label-specific fluorescent reporter probes were:            Probe123: 5′6-FAM/GCCTGTAGTCCCACGCGATCTAACA/3′BHQ_1 for            detection of label 2OS-A1B1 and Probe124:            5′HEX/CAACCATTGATTGGGGACAACTGGG/3′BHQ_1 for detection of            label 2OS-A2B2. The label specific reporter probes were            purchased from DNA Technology (Denmark) and delivered as            lyophilized powder. Stock dilutions of 100 μM            oligonucleotides were made in nuclease free water and stored            at −20° C. Primers used for amplification in this experiment            forward: GAAGTTCCAGCCAGCGTC, and reverse:            CTGTGACTATGTGAGGCTTTC.        -   b. Analysis: combined with the above.

Example 1—Results and Conclusions

After sorting of PE labeled cells and QPCR the resultant Ct valuesconfirmed that Detection Molecules were enriched, i.e. 2OS DNA-barcodeLabels were successfully recovered, only when associated with the CMVepitope, while they were not detected when associated with the HIVepitope (FIG. 10 ). This was observed even when labels were invertedbetween the two Binding Molecules, implying that the recovery was notLabel specific, but truly specific for the Binding molecule.

It was verified that the 2OS labels were recovered after cellularinteraction, sorting and QPCR only if they were associated with positivecontrol Detection molecules, and not if they were associated withnegative control Detection molecules.

Example 2

In this example the binding molecules are pMHC complexes, the linker isa dextran-streptavidin-fluorochrome conjugate, and the label is a DNAoligonucleotide.

The sample is a HPBMC, the isolating and/or detecting is by FACS, andthe determining of the identity of the label is by QPCR.

Example 2 Explained

This is an example where the Sample (1) was blood from one CMV positiveand HIV negative donor which was modified (1b) to generate Peripheralblood mononuclear cells (PBMCs).

The Linker (2) was a dextran conjugate with streptavidin andfluorochrome (Dextramer backbone from Immudex).

The example is similar to example 1 except that a 1000 fold excess ofDetection Molecules with irrelevant Binding Molecules but without labelwere included. The Binding Molecules used (3) are peptide-MHC (pMHC)complexes displaying either CMV (positive antigen) or HIV (negativeantigen) derived peptide-antigens or pMHC complexes displayingirrelevant peptide antigen. The Binding Molecules were modified (3b) bybiotinylation to provide a biotin capture-tag for the Linker. Thebinding Molecules were purified (2c) by HPLC.

The Labels (4) were single stranded oligonucleotides applied asDNA-barcodes. The oligonucleotides were synthetized (4a) by DNATechnology A/S (Denmark) and were synthetically modified (4b) with aterminal biotin capture-tag.

The Detection Molecule (5) was synthetized (5a) by attaching bindingmolecules in the form of biotinylated pMHC and Labels in the form ofbiotin-modified oligonucleotides, DNA-barcodes, onto astreptavidin-modified dextran linker. The detection molecule furthercontained a modification (5b) in the form of a fluorochrome. Threedifferent detection molecules were generated wherein the two of theseindividual detection molecules containing CMV- and HIV-directed pMHCwere encoded for by corresponding individual DNA-barcodes. DetectionMolecules with irrelevant Binding Molecules (p*MHC) were not encoded forwith a DNA-barcode.

An amount of sample, PBMC's (1b) was incubated with an amount of mixeddetection molecules (5) in a ratio of 1:1:998(Labeled-CMV:Labeled-HIV:unlabeled-irrelevant directed BindingMolecules) under conditions (6c) that allowed binding of detectionmolecules to T cells in the sample.

The cell-bound detection molecules were separated from the non-cellbound detection molecules (7) by first a few rounds of washing thePBMC's through centrifugation sedimentation of cells and resuspension inwash buffer followed by Fluorescence Activated Cell Sorting (FACS) offluorochrome labeled cells. T cells that can efficiently bind detectionmolecules will fluoresce because of the fluorochrome comprised withinthe detection molecules; T cells that cannot bind detection moleculeswill not fluoresce. FACS-sorting leads to enrichment of fluorescentcells, and hence, enrichment of the detection molecules along with theassociated labels that bind T cells of the PBMC sample.

FACS isolated cells were subjected to quantitative PCR analysis of theoligonucleotide label associated with the detection molecules bound tothe isolated cells to reveal the identity of the detection moleculesthat bound to the T cells present in the sample. This example thusrevealed the presence of T cells in the blood expressing a T cellreceptor that binds to peptide-MHC molecules represented within thelibrary of Detection Molecules. It also revealed the feasibility ofenriching for Detection Molecules based on the presence of T cellsspecific for the positive peptide-antigen (CMV) over the negativepeptide-antigen (HIV) antigens. In this setting, it was proved that itwas possible to specifically enrich Detection Molecules associated withpositive peptide-antigen when excess of irrelevant Detection Moleculewas included in the sample incubation.

Example 2 in Detail

-   -   1. Sample preparation. The cell sample used in this example was        obtained in the same way as described in example 1    -   2. Linker preparation: The linker used in this example was        prepared as in example 1    -   3. Binding Molecules preparation: The binding molecules used in        this example were two different class I MHC-peptide complexes.        MHC heavy chains (HLA-A02 and HLA-B07) and B2M were expressed in        E.coli as previously described (Hadrup et al. 2009) and each        MHC-I complex generated with two peptide antigens or left with        an irrelevant photolabile 9-mer peptide. The individual        specificities (allele and peptide combination) were generated in        the following way:        -   a. Synthesis: As in example 1.            -   i. HIV derived peptide ILKEPVHGV from antigen HIV                polymerase and CMV derived peptide TPRVTGGGAM from                antigen pp65 TPR (Pepscan Presto, NL) were diluted in                phosphate buffered saline (DPBS; Lonza) and mixed to                final concentrations 100 μg/ml:200 μM (HLA-A0201:                ILKEPVHGV and HLA-B0702:TPRVTGGGAM). The mixtures were                exposed to 366 nm UV light (UV cabinet; CAMAG) for one                hour and optionally stored for up to 24 h at 4° C. The                irrelevant Binding Molecules, p*HLA-A0201 and                p*HLA-B0702 were mixed in equal amounts (pg/ml) and                diluted to 100 μg/ml in DPBS.        -   b. Modification: No further modifications        -   c. Purification: Before applying the peptide exchanged HLA            monomer Binding Molecules for preparation of detection            molecules these were centrifuged 5 min, 3300 g, to sediment            any aggregated MHC molecules. The p*MHC monomers were not            purified further.    -   4. Label preparation: In this example Labels were synthetic        oligonucleotides modified with biotin for coupling to the        Linker.        -   a. Synthesis: The Labels were oligonucleotides, DNA            barcodes, which were purchased from DNA Technology (Denmark)            and delivered as lyophilized powder. Stock dilutions of 100            μM oligonucleotide were made in nuclease free water and            stored at −20° C.            -   i. The Label system used in this example was named 1OS                and comprised of single stranded oligonucleotides which                were applied as a DNA barcodes (Oligo 4:                5GAGATACGTTGACCTCGTTGAANNNNNNTCTTGAACTATGA                ATCGTCTCACTTAAGCTCTTGGTTGCAT and Oligo 5:                5GAGATACGTTGACCTCGTTGAANNNNNNTCTATAGGTGTC                TACTACCTCACTTAAGCTCTTGGTTGCAT were used in this                experiment, 5 indicates a 5′ biotin modification).        -   b. Modification: All Labels were diluted to working            concentrations (2.17 uM) in nuclease free water with 0.1%            Tween and stored at −20° C.        -   c. Purification: Labels were not purified further.    -   5. Detection Molecules preparation: The Binding Molecules        (pMHCs) and Labels (oligonucleotides) were attached to the        Linker (dextran-streptavidin-PE conjugate), to form Detection        Molecules, in such a way that a given pMHC was always attached        to a given DNA-barcode.        -   a. Synthesis: In this example the Detection Molecules were            prepared by attaching 1OS DNA-barcodes prior to addition of            pMHC. Detection Molecules were essentially generated in the            same way as described in example 1, with the difference that            two different sets of two detection molecules were            generated.            -   Each set had two specificities individually labeled. The                Labels were inverted between the two sets as described                below. Moreover a third Detection Molecule was generated                without any Label, but comprised of the Linker and a                Binding Molecule (p*MHC), this detection molecule was                included in both sets of Detection Molecules as                described below in the indicated stoichiometry:            -   1. 1×CMV specific pMHCs (HLA-A0201: ILKEPVHGV) coupled                to Oligo4, 1×HIV specific pMHCs (HLA-B0702:TPRVTGGGAM)                coupled to Oligo5 and 998× non-labeled p*MHC                (HLA-A0201:p* and HLA-B0702:p*).                -   2. 1×CMV specific pMHCs (HLA-A0201: ILKEPVHGV)                    coupled to Oligo5, 1×HIV specific pMHCs                    (HLA-B0702:TPRVTGGGAM) coupled to Oligo4 and 998×                    non-labeled p*MHC (HLA-A0201:p* and HLA-B0702:p*).        -   b. Modification: Since the total volume of each set of            Detection Molecules exceeded 100 μl this volume was reduced            to reach a desired concentration of specific Binding            Molecules in the pooled solution of Detection Molecules.            -   i. Size exclusion spin columns (Nanosep 300K Omega, Pall                Corporation) with a cut-off at 300 kDa were saturated by                adding 500 μl 2% BSA/DPBS and centrifuging 5000 g, until                the volume had passed through. Subsequently, the columns                were washed twice by adding 500 μl DPBS and centrifuging                5000 g until no volume was left in the columns. Each                pooled set of detection molecules were added to a spin                column and centrifuged 5000 g, 4° C., until the desired                volume resided in the column (80 μl per incubation with                sample). The reduced volume of each set of Detection                Molecules was moved to new containers.        -   c. Purification: The Detection Molecules were centrifuged 5            min, 3300 g, to sediment any aggregates before being added            to the sample.    -   6. Incubation of sample and Detection Molecules: The cell sample        and the Detection Molecules were incubated in the same way as        described in example 1.    -   7. Enrichment of Detection Molecules with desired        characteristics: The Detection Molecules were enriched for in        the same way as described in example 1.    -   8. Identification of enriched Detection Molecules: Enriched        Detection Molecules were essentially identified in the same way        as described in example 1, with the difference that two        different label-specific fluorescent reporter probes were        applied: LNA-4:        5′6-FAM/TCT[+T][+G][+A]AC[+T][+A]TG[+A][+A][+T]CGTC/3′BHQ-1-plus        for detection of Oligo4 and LNA-5:        5′HEX/TCT[+A][+T][+A]GG[+T][+G]TC[+T][+A][+C]TACC/3′BHQ-1-plus        for detection of Oligo5 ([+X] indicating locked nucleic acids        (LNAs)). Primers used for amplification of Oligo 4 and Oligo 5,        forward: GAGATACGTTGACCTCGTTG and reverse: ATGCAACCAAGAGCTTAAGT.

Example 2—Results and Conclusions

After sorting of PE labeled cells and QPCR the resultant Ct valuesconfirmed that Detection Molecules were enriched, i.e. 1OS DNA-barcodeLabels were successfully recovered, only when associated with the CMVepitope, while they were not detected when associated with the HIVepitope (FIG. 11 ). This was observed even when labels were invertedbetween the two Binding Molecules, implying that the recovery was notLabel specific, but truly specific for the Binding molecule. Moreoverthe example demonstrated that a great amount of irrelevant Detectionmolecule equipped with the same fluorescent label as all Detectionmolecules in the incubation would not be detrimental for enrichment ofcells that would fluoresce due to specific binding with a Detectionmolecule.

It was verified that the 1OS Labels were recovered after cellularinteraction, sorting and QPCR only if they were associated with positivecontrol Detection molecules, and not if they were associated withnegative control Detection molecules, even in the presence of a highamount of irrelevant Detection molecule.

Example 3

In this example the binding molecules are pMHC representing 6 differentHLA-alleles, the linker is dextran-streptavidin-PE conjugate and thelabel is a DNA oligonucleotide.

The sample is HPBMC, the isolating and/or detecting is by FACS, and thedetermining of the identity of the label is by sequencing.

Example 3 Explained

This is an example where the Samples (1) were blood from one donor thatwere HLA-B0702:CMV pp65 TPR positive and another donor that wereHLA-B0702 negative which were modified (1b) to generate Peripheral bloodmononuclear cells (PBMCs). These samples were mixed in different ratiosto generate new samples with different but known frequencies of T cellsspecific toward the HLA-B0702:CMV epitope.

The Linker (2) was a dextran conjugate with streptavidin andfluorochrome (Dextramer backbone from Immudex).

The Binding Molecules (3) were peptide-MHC (pMHC) complexes displayingone out of 110 different peptide-antigens comprised within 6 differentHLA-types. The MHC molecules were modified (3b) by biotinylation toprovide a biotin capture-tag for the Linker. The binding molecules werepurified (2c) by HPLC and quality controlled in terms of the formationof functional pMHC multimers for staining of control T-cell populations.

The Labels (4) were single stranded oligonucleotide applied asDNA-barcodes. The oligonucleotides were synthetized (4a) by DNATechnology A/S (Denmark) and were synthetically modified (4b) with aterminal biotin capture-tag.

The Detection Molecule (5) was synthetized (5a) by attaching BindingMolecules in the form of biotinylated pMHC and Labels in the form ofbiotin-modified oligonucleotides (DNA-barcodes) onto astreptavidin-modified dextran linker. The detection molecule furthercontained a modification (5b) in the form of a fluorochrome. A libraryof 110 different Detection Molecules were generated wherein individualBinding Molecules, comprised of different pMHC, were encoded for bycorresponding individual Labels, comprised of different DNA-barcodes.

An amount of sample, PBMC's (1b) was incubated with an amount of mixedDetection Molecules (5) under conditions (6c) allowing binding ofDetection Molecules to T cells in the sample.

The cell-bound Detection Molecules were separated from the non-cellbound Detection Molecules (7) by first a few rounds of washing thePBMC's through centrifugation sedimentation of cells and resuspension inwash buffer followed by Fluorescence Activated Cell Sorting (FACS) offluorochrome labeled cells. T cells that can efficiently bind DetectionMolecules will fluoresce because of the fluorochrome comprised withinthe detection molecules; T cells that cannot bind detection moleculeswill not fluoresce. FACS-sorting leads to enrichment of fluorescentcells, and hence, enrichment of the detection molecules along with theassociated labels that bind T cells of the PBMC sample.

FACS isolated cells were subjected to PCR for specific amplification ofthe DNA-barcode Label associated with the Detection Molecules bound tothe isolated cells. High throughput sequencing of the resultant PCRproduct revealed the identity of Detection Molecules that bound to Tcells present in the sample.

This example thus revealed the presence of T cells in the bloodexpressing a T cell receptor that binds to pMHC molecules representedwithin the library of Detection Molecules. The number of sequencingreads mapped to a given DNA-barcode and its corresponding BindingMolecule would mirror the frequency of the T cells found by conventionalMHC multimer stainings using the same Binding Molecule.

Example 3 in Detail

-   -   1. Sample preparation. The cell samples used in this example was        obtained by preparing PBMC's from blood drawn from one donor        that were HLA-B0702:CMV pp65 TPR positive and from another donor        that were HLA-B0702 negative, as determined by conventional        pMHC-multimer and antibody stainings.        -   a. Acquiring sample: As in example 1        -   b. Modifying sample: PBMCs were isolated from whole blood as            described in example 1.            -   i. Mixing of PBMCs from the two donors provided a                titration of the HLA-B0702 CMV pp65 TPR responses in a                B0702 negative donor sample. 5 fold dilutions of BC260                into BC262 were applied to generate seven samples with:                100, 20, 4, 0.8, 0.16, 0.032 and 0.0064% of cells                derived from BC260. This corresponded to theoretical                frequencies of HLA-B0702 CMV pp65 TPR specific T cells                of 5%, 1%, 0.2%, 0.04%, 0.008%, 0.0016% and 0.00032%.                The samples were in turn applied to evaluate the                sensitivity of the Detection Molecules for detecting                antigen-specific T cells in a sample. The relevance of                the results obtained after applying the Detection                Molecules could be evaluated by comparison of results                obtained by conventional pMHC-multimer staining when                applying a corresponding sample.    -   2. Linker preparation: The linker used in this example was        prepared as in example 1-2.    -   3. Binding Molecules preparation: The Binding Molecules used in        this example were class I MHC-peptide complexes. The individual        specificities (allele and peptide combination) were generated as        described in example 1. A library of 110 different pMHCs,        comprised of 6 different HLA-types, were generated, these are        listed in table 9.        -   a. Synthesis: As described in example 1-2        -   b. Modification: No further modifications        -   c. Purification: As described in example 1-2    -   4. Label preparation: In this example Labels were synthetic        oligonucleotides modified with biotin for coupling to the        Linker. 110 different Labels from the 1OS system were applied        (table 8).        -   a. Synthesis: As described in example 2            -   i. The Label system used in this example were named 1OS                and comprised of single stranded oligonucleotides which                were applied as a DNA barcodes.        -   b. Modification: As described in example 1-2.        -   c. Purification: As described in example 1-2.    -   5. Detection Molecules preparation: 110 different Detection        Molecules were generated, each with a different Binding Molecule        encoded by a unique Label. The Binding Molecules (pMHCs) and        Labels (1OS DNA-barcodes) were attached to the Linker        (dextran-streptavidin-PE conjugate), to form Detection        Molecules, in such a way that a given pMHC was always attached        to a given DNA-barcode.        -   a. Detection Molecules were essentially generated in the            same way as described in example 2, with the difference that            110 different Detection Molecules were generated. The given            combination of Label (DNA-barcode) and Binding Molecule            (pMHC) of each Detection Molecule are presented in table 9.        -   b. Modification: Since the total volume of 110 pooled            Detection Molecules exceeded 100 μl this volume was reduced            to reach a desired concentration of specific Binding            Molecules. This was done as described in example 2        -   c. Purification: As described in example 1-2.    -   6. Incubation of sample and Detection Molecules: The cell sample        and the Detection Molecules were mixed in one container to allow        Detection Molecules to bind to T cells.        -   a. Amount of sample: Duplicates of seven samples each            comprised of 2×10E6 cells in the form of BC's, i.e.            2×(1×100% BC260 and 6× five-fold dilutions of BC260 into            BC262) (as described in 1.b)        -   b. Amount of Detection Molecule: As described in example 1        -   c. Conditions: BC260 and BC262 were thawed individually in            10 ml, 37° C., RPMI with 10% fetal bovine serum (FBS),            centrifuged 5 min, 490 g, and washed twice in 10 ml RPMI            with 10% FBS. All washing of cells refer to centrifugation 5            min, 490 g, with subsequent removal of supernatant. BC's            were incubated individually in 50 nM dasatinib, 30 min,            37° C. and resuspended in 10 ml per 2×10E6 cells of the            respective BC. Five-fold dilutions of BC260 were produced by            adding 2.5 ml (0.5×10E6) of the former cell sample into 10            ml (2×10E6 cells) of the BC262 sample. The mixed cellular            samples were washed in 200 ul barcode-buffer (PBS/0.5% BSA/2            mM EDTA/100 μg/ml herring DNA) and resuspended in this            buffer to approximately 20 μl per sample prior to incubation            of cells with Detection Molecules.            -   i. The Detection molecules were added in the required                amount and samples were incubated as described in                example 1-2.    -   7. Enrichment of Detection Molecules with desired        characteristics: The Detection Molecules were enriched for in        the same way as described in example 1-2.    -   8. Identification of enriched Detection Molecules: Because the        Detection Molecules were enriched based on specific binding of        the Binding Molecule (the pMHC) to cells, the identification of        the associated Labels (DNA-barcodes) amongst the sorted cells        would also reveal the pMHCs that had bound to cells in the PBMC        sample. In this example the DNA-barcodes associated with the        enriched Detection Molecules, were amplified by PCR and        identified by high-throughput sequencing.        -   a. Apply. The sorted cell sample which contained            DNA-barcodes derived from the enriched Detection Molecules            were amplified by PCR. See table 10 for composition of the            PCR and table 11 for the thermal profile. The Taq PCR Master            Mix Kit (Qiagen, #201443) was applied and PCR was run on the            thermal cycler: GeneAmp, PCR System 9700 (Applied            Biosystem). PCR products were visualized after gel            electrophoresis on a Bio-Rad Gel Doc EZ Imager. DNA was            sequenced using the Ion Torrent PGM platform (Life            Technologies)            -   i. Primers were purchased from DNA Technology (Denmark)                and delivered as lyophilized powder. Stock dilutions of                100 μM were made in nuclease free water and stored at                −20° C. The primers included adaptors for Ion Torrent                sequencing, i.e. an A-key and a P1-key on the forward                and reverse primer respectively. Additionally the                forward primers had unique DNA sequences besides the                primer region and the A-key (the primer sequences are                listed in table 12). These primers were used to assign                DNA-barcodes derived from the same sample with a                sample-identification sequence (Sample-ID barcode) (see                FIG. 9 for a schematic presentation of this design).                This enabled distribution of DNA-barcode sequence reads                according to their originating sample, when DNA-barcodes                from multiple samples were sequenced in the same                sequencing reaction. The non-enriched library of the 110                different Detection Molecules (diluted 100.000× after                being reduced in volume) were also assigned with a                sample-ID barcode through PCR (referred to as the                Detection Molecule input). Information about the                distribution of Labels within the library of Detection                Molecules before enrichment would allow normalization of                the sequence output. Pooled PCR products derived from                the sample input and from multiple incubations of                Detection Molecule and sample were purified with the                MinElute PCR purification Kit (Qiagen, #28006) according                to standard procedure.            -   ii. Purified DNA was sequenced by GeneDx (U.S.A) on an                Ion Torrent PGM 314 chip.        -   b. Analysis. Positive sequence reads were aligned to            sequences that read from the sample-barcode-identity at the            5′-end all the way through the DNA-barcode-identity. The            number of reads was normalized according to the total number            of reads that mapped to the same sample-ID barcode and            according to the Detection Molecule input reads.            -   i. Mapping sequencing reads to 1OS DNA-barcodes: A                sequence database was created consisting of the possible                combinations of 15 sample-identification barcodes and                110 1OS DNA-barcodes together with the primer sequences                from the 1OS system. This accumulated to 1650 sequences                that could be expected from a sequencing run. Each                sequencing read was then used to search the database for                alignments, using the nucleotide BLAST algorithm, with a                match reward of 1, mismatch reward of −2 and a gap cost                of 2 for both opening and extending a gap. In this way                sequencing errors were penalized equally, whether a base                was miscalled or inserted/deleted in the sequencing read                compared to the actual sequence. Alignments were                discarded by the following criteria:                -   1. E-value >1e-12; insufficient length of alignment                    (should be greater than 60 for the 1OS barcodes).                -   2. Start position in subject sequence larger than 2,                    i.e. fewer than 5 out of 6 bases in the unique part                    of the sample-identification barcode was included in                    the alignment.            -   ii. If multiple alignments could still be found for any                sequencing read, only the alignment with the best                percent identity was kept. Finally, the number of reads                mapping to each DNA barcode in the database was counted.            -   iii. Identifying overrepresented DNA barcodes: Relative                read counts were calculated by normalizing each read to                the total read count mapping to the same sample-ID                barcode. The relative read counts were then used to                calculate the fold change per DNA-barcode compared to                the control DNA-barcode Detection Molecule input (the                non-enriched Detection Molecule library). Significantly                overrepresented DNA-barcodes were identified using a                2-sample test for equality of proportions on the raw                read counts in a sample versus the DNA-barcode                input-sample, and p-values were corrected for multiple                testing using the Benjamini-Hochberg FDR method.

Example 3—Results and Conclusions

FACS sorting of fluorescent labeled cells, specific amplification ofDNA-barcode Labels and high-throughput sequencing verified that it waspossible to enrich and detect 1OS barcodes from a library of multipledifferent Detection molecules composed of 110 different 2OS DNA-barcodeLabels encoding for 110 different antigen specificities distributed on 6different HLA-types (FIG. 12 ). Moreover the number of sequence readsrecovered from a given 1OS barcode was sensitive to the frequency ofantigen specific T cells in the sample.

This example demonstrates that it is possible to detect antigen specificT cell responses of different frequencies in a panel of 110 different1OS labeled Detection Molecules.

Example 4

In this example the binding molecules are class I pMHC complexescomprising 6 different HLA alleles, the linker isdextran-streptavidin-PE conjugate and the label is a DNAoligonucleotide.

The sample is HPBMC, the isolating and/or detecting is by FACS, and thedetermining of the identity of the label is by sequencing.

Example 4 Explained

This example was essentially the same as example 3 only another Labelsystem was applied.

This is an example where the Samples (1) were blood from one donor thatwere HLA-B0702:CMV pp65 TPR positive and another donor that wereHLA-B0702 negative which were modified (1b) to generate Peripheral bloodmononuclear cells (PBMCs). These samples were mixed in different ratiosto generate new samples with different but known frequencies of T cellsspecific toward the HLA-B0702:CMV epitope. The Linker (2) was a dextranconjugate with streptavidin and fluorochrome (Dextramer backbone fromImmudex).

The Binding Molecules (3) were peptide-MHC (pMHC) complexes displayingone out of 110 different peptide-antigens comprised within 6 differentHLA-types. The MHC molecules were modified (3b) by biotinylation toprovide a biotin capture-tag for the Linker. The binding molecules werepurified (2c) by HPLC and quality controlled in terms of the formationof functional pMHC multimers for staining of control T-cell populations.

The Labels (4) were oligonucleotides applied as DNA-barcodes. Theoligonucleotides were synthetized (4a) by DNA Technology A/S (Denmark)and were synthetically modified (4b) with a terminal biotin capture-tag.The labels were combined oligonucleotide labels arising by annealing anA oligonucleotide (modified with biotin) to a partially complimentary Boligonucleotide label followed by enzymatic DNA polymerase extension ofOligo A and Oligo B to create a fully double stranded label. TheDetection Molecule (5) was synthetized (5a) by attaching BindingMolecules in the form of biotinylated pMHC and Labels in the form ofbiotin-modified oligonucleotides (DNA-barcodes) onto astreptavidin-modified dextran linker. The detection molecule furthercontained a modification (5b) in the form of a fluorochrome. A libraryof 110 different Detection Molecules were generated wherein individualBinding Molecules, comprised of different pMHC, were encoded for bycorresponding individual Labels, comprised of different DNA-barcodes.

An amount of sample, PBMC's (1b) was incubated with an amount of mixedDetection Molecules (5) under conditions (6c) allowing binding ofDetection Molecules to T cells in the sample.

The cell-bound Detection Molecules were separated from the non-cellbound Detection Molecules (7) by first a few rounds of washing thePBMC's through centrifugation sedimentation of cells and resuspension inwash buffer followed by Fluorescence Activated Cell Sorting (FACS) offluorochrome labeled cells. T cells that can efficiently bind DetectionMolecules will fluoresce because of the fluorochrome comprised withinthe detection molecules; T cells that cannot bind detection moleculeswill not fluoresce. FACS-sorting leads to enrichment of fluorescentcells, and hence, enrichment of the detection molecules along with theassociated labels that bind T cells of the PBMC sample.

FACS isolated cells were subjected to PCR for specific amplification ofthe DNA-barcode associated with the Detection Molecules bound to theisolated cells. High throughput sequencing of the resultant PCR productrevealed the identity of Detection Molecules that bound to T cellspresent in the sample.

This example thus revealed the presence of T cells in the bloodexpressing a T cell receptor that binds to pMHC molecules representedwithin the library of Detection Molecules. The number of sequencingreads mapped to a given DNA-barcode and its corresponding BindingMolecule would mirror the frequency of the T cells found by conventionalMHC multimer stainings using the same Binding Molecule. Moreover itrevealed that Labels, i.e. Detection Molecules, associated with T cellsin a sample would be sufficiently enriched to reveal the presence of lowfrequent T cells binding such Detection Molecules.

Example 4 in Detail

-   -   1. Sample preparation. The cell samples used in this example        were obtained by preparing PBMC's from blood drawn from one        donor that were HLA-B0702:CMV pp65 TPR positive and from another        donor that were HLA-B0702 negative, as determined by        conventional pMHC-multimer and antibody staining. They were        acquired (a.) and modified (b.) in the same way as described in        example 3.    -   2. Linker preparation: The linker used in this example was        prepared as in example 1-3.    -   3. Binding Molecules preparation: The Binding Molecules used in        this example were class I MHC-peptide complexes. The individual        specificities (allele and peptide combination) were generated as        described in example 1-3. A library of 110 different pMHCs were        generated, comprised of 6 different HLA-types, these are listed        in table 9.        -   a. Synthesis: As described in example 1-3        -   b. Modification: No further modifications        -   c. Purification: As described in example 1-3    -   4. Label preparation: In this example Labels were synthetic        oligonucleotides modified with biotin for coupling to the        Linker. 110 different Labels from the 2OS system were applied        (table 2).        -   a. Synthesis: As described in example 1.            -   i. The Label system used in this example was named 2OS                and was developed to increase the complexity of a                limited number of oligonucleotide sequences. This was                enabled by applying a combinatorial strategy where two                partially complementary oligonucleotides (an A                oligonucleotide with a 5′ biotin tag and a B                oligonucleotide) where annealed and then elongated to                produce new unique oligonucleotide-sequences (AxBy)                which were applied as a DNA-barcodes (Labels) (FIG. 9 ).                By combining 6 unique oligonucleotide-sequences (A label                precursor) that were all partly complementary to 20                other unique oligonucleotide sequences (B label                precursor) a combinatorial library of 120 different                (AxBy) Labels were produced. Only 110 of these Labels                were used in this example (table 9).        -   b. Modification: As described in example 1-3.        -   c. Purification: As described in example 1-3.    -   5. Detection Molecules preparation: 110 different Detection        Molecules were generated, each with a different Binding Molecule        encoded by a unique Label. The Binding Molecules (pMHCs) and        Labels (2OS DNA-barcodes) were attached to the Linker        (dextran-streptavidin-PE conjugate), to form Detection        Molecules, in such a way that a given pMHC was always attached        to a given DNA-barcode.        -   a. Detection Molecules were essentially generated in the            same way as described in example 3. The given combination of            Label (2OS DNA-barcode) and Binding Molecule (pMHC) of each            Detection Molecule are presented in table 9.        -   b. Modification: Since the total volume of 110 pooled            Detection Molecules exceeded 100 μl this volume was reduced            to reach a desired concentration of specific Binding            Molecules. This was done as described in example 2-3.        -   c. Purification: As described in example 1-3.    -   6. Incubation of sample and Detection Molecules: The cell sample        and the Detection Molecules were mixed in one container to allow        Detection Molecules to bind to T cells.        -   a. Amount of sample: Samples were equivalent to those used            in example 3.        -   b. Amount of Detection Molecule: As described in example 1-3        -   c. Conditions: Samples and Detection Molecules were treated            under the same conditions as described in example 3.    -   7. Enrichment of MHC molecules with desired characteristics: The        Detection Molecules were enriched for in the same way as        described in example 1-3.    -   8. Identification of enriched Detection Molecules: Because the        Detection Molecules were enriched based on specific interaction        of the Binding Molecule (the pMHC) with cells, the        identification of the associated Labels (DNA-barcodes) amongst        the sorted cells would also reveal the pMHCs that had bound to        cells in the PBMC sample. In this example the DNA-barcodes        associated with the enriched Detection Molecules, were amplified        by PCR and identified by high-throughput sequencing.        -   a. Apply. The sorted cell sample which contained            DNA-barcodes derived from the enriched Detection Molecules            were amplified by PCR. See table 10 for composition of the            PCR and table 11 for the thermal profile. The Taq PCR Master            Mix Kit (Qiagen, #201443) was applied and PCR was run on the            thermal cycler: GeneAmp, PCR System 9700 (Applied            Biosystem). PCR products were visualized after gel            electrophoresis on a Bio-Rad Gel Doc EZ Imager. DNA was            sequenced using the Ion Torrent PGM platform (Life            Technologies)            -   i. Primers were purchased from DNA Technology (Denmark)                and delivered as lyophilized powder. Stock dilutions of                100 μM were made in nuclease free water and stored at                −20° C. The primers included adaptors for Ion Torrent                sequencing, i.e. an A-key and a P1-key on the forward                and reverse primer respectively. Additionally the                forward primers had unique DNA sequences besides the                primer region and the A-key. These primers were used to                assign DNA-barcodes derived from the same sample with a                sample-identification sequence (Sample-ID barcode) (the                primer sequences are listed in table 13). This enabled                distribution of DNA-barcode sequence reads according to                their originating sample, when DNA-barcodes from                multiple samples were sequenced in the same sequencing                reaction. The non-enriched library of the 110 different                Detection Molecules (diluted 100.000× after being                reduced in volume) were also assigned with a sample-ID                barcode through PCR (referred to as the Detection                Molecule input)(see FIG. 9 for a schematic overview of                the primer design). Information about the distribution                of Labels within the library of Detection Molecules                before enrichment would allow normalization of the                sequence output. Pooled PCR products derived from the                sample input and from multiple incubations of Detection                Molecule and sample were purified with the MinElute PCR                purification Kit (Qiagen, #28006) according to standard                procedure.            -   ii. The purified DNA was sequenced by GeneDx (U.S.A) on                an Ion Torrent PGM 314 chip.        -   b. Analysis. Positive sequence reads were aligned to            sequences that read from the sample-barcode-identity at the            5′-end all the way through the DNA-barcode-identity. The            number of reads was normalized according to the total number            of reads that mapped to the same sample-ID barcode and            according to the Detection Molecule input reads. The            Analysis was essentially as in example 3 except that another            database of 2OS sequences was generated and sequences were            mapped according to corresponding 2OS DNA-barcodes.            -   i. Mapping sequencing reads to 2OS DNA-barcodes: A                sequence database was created consisting of the possible                combinations of 15 sample-identification barcodes and                120 2OS DNA barcodes (together with the primer and                annealing sequences from the 2OS system). This                accumulated to 1800 sequences that could be expected                from a sequencing run. Each sequencing read was then                used to search the database for alignments, using the                nucleotide BLAST algorithm, with a match reward of 1,                mismatch reward of −2 and a gap cost of 2 for both                opening and extending a gap. In this way sequencing                errors were penalized equally, whether a base was                miscalled or inserted/deleted in the sequencing read                compared to the actual sequence. Alignments were                discarded by the following criteria:                -   1. E-value >1e-12; insufficient length of alignment                    (should be greater than 102 for the 2OS barcodes).                -   2. Start position in subject sequence larger than 2,                    i.e. fewer than 5 out of 6 bases in the unique part                    of the sample-identification barcode was included in                    the alignment.            -   ii. If multiple alignments could still be found for any                sequencing read, only the alignment with the best                percent identity was kept. Finally, the number of reads                mapping to each DNA barcode in the database was counted.            -   iii. Identifying overrepresented DNA barcodes: Relative                read counts were calculated by normalizing each read to                the total read count mapping to the same sample-ID                barcode. The relative read counts were then used to                calculate the fold change per DNA-barcode compared to                the control DNA-barcode Detection Molecule input (the                non-enriched Detection Molecule library). Significantly                overrepresented DNA-barcodes were identified using a                2-sample test for equality of proportions on the raw                read counts in a sample versus the DNA-barcode                input-sample, and p-values were corrected for multiple                testing using the Benjamini-Hochberg FDR method.

Example 4—Results and Conclusions

FACS sorting of fluorescent labeled cells, specific amplification ofDNA-barcode Labels and high-throughput sequencing verified that it waspossible to enrich and detect 2OS barcodes from a library of multipledifferent Detection molecules composed of 110 different 2OS DNA-barcodeLabels encoding for 110 different antigen specificities distributed on 6different HLA-types (FIG. 13 ). Moreover the number of sequence readsrecovered from a given 2OS barcode was sensitive to the frequency ofantigen specific T cells in the sample, also indicating that it will bepossible to detect Labels associated with responses of very lowfrequency (<0.002).

This example demonstrates that it is possible to detect antigen specificT cell responses of different and of low frequencies in a panel of 110different 2OS labeled Detection Molecules.

Example 5

In this example the binding molecules are class I pMHCs, the linker isstreptavidin conjugate and the label is DNA.

The isolating and/or detecting is by FACS, and the determining of theidentity of the label is by sequencing.

Example 5 Explained

This is an example where the Samples (1) were blood from six differentdonors with different HLA-types which were modified (1b) to generatePeripheral blood mononuclear cells (PBMCs).

The Linker (2) was a dextran conjugate with streptavidin andfluorochrome (Dextramer backbone from Immudex).

The Binding Molecules (3) were peptide-MHC (pMHC) complexes displayingone out of 110 different peptide-antigens comprised within 6 differentHLA-types. The MHC molecules were modified (3b) by biotinylation toprovide a biotin capture-tag for the Linker. The binding molecules werepurified (2c) by HPLC and quality controlled in terms of the formationof functional pMHC multimers for staining of control T-cell populations.

The Labels (4) were single stranded oligonucleotide applied asDNA-barcodes. The oligonucleotides were synthetized (4a) by DNATechnology A/S (Denmark) and were synthetically modified (4b) with aterminal biotin capture-tag.

The Detection Molecule (5) was synthetized (5a) by attaching BindingMolecules in the form of biotinylated pMHC and Labels in the form ofbiotin-modified oligonucleotides (DNA-barcodes) onto astreptavidin-modified dextran linker. The detection molecule furthercontained a modification (5b) in the form of a fluorochrome. A libraryof 110 different Detection Molecules were generated wherein individualBinding Molecules, comprised of different pMHC, were encoded for bycorresponding individual Labels, comprised of different DNA-barcodes.

An amount of sample, PBMC's (1b) was incubated with an amount of mixedDetection Molecules (5) under conditions (6c) allowing binding ofDetection Molecules to T cells in the sample.

The cell-bound Detection Molecules were separated from the non-cellbound Detection Molecules (7) by first a few rounds of washing thePBMC's through centrifugation sedimentation of cells and resuspension inwash buffer followed by Fluorescence Activated Cell Sorting (FACS) offluorochrome labeled cells. T cells that can efficiently bind DetectionMolecules will fluoresce because of the fluorochrome comprised withinthe detection molecules; T cells that cannot bind detection moleculeswill not fluoresce. FACS-sorting leads to enrichment of fluorescentcells, and hence, enrichment of the detection molecules along with theassociated labels that bind T cells of the PBMC sample.

FACS isolated cells were subjected to PCR for specific amplification ofthe DNA-barcode associated with the Detection Molecules bound to theisolated cells. High throughput sequencing of the resultant PCR productrevealed the identity of Detection Molecules that bound to T cellspresent in the sample.

This example thus revealed the presence of T cells in the bloodexpressing a T cell receptor that binds to pMHC molecules representedwithin the library of Detection Molecules. The application of multiple(110) Detection Molecules in one sample enabled detection of T cellswith different T cell receptor specificities in parallel.

Example 5 in Detail

-   -   1. Sample preparation. The cell samples used in this example        were obtained by preparing PBMC's from blood drawn from six        different donors with a number of different peptide-antigen        responsive T cells, as determined by conventional pMHC-multimer        staining.        -   a. Acquiring sample: Blood was obtained from the Danish            Blood Bank, as example 1-4        -   b. Modifying sample: PBMCs were isolated from whole blood as            described in example 1-4.    -   2. Linker preparation: The linker used in this example was        prepared as in example 1-4.    -   3. Binding Molecules preparation: The Binding Molecules used in        this example were class I MHC-peptide complexes. The individual        specificities (allele and peptide combination) were generated as        described in example 1. A library of 110 different pMHCs,        comprised of 6 different HLA-types, were generated, these are        listed in table 9.        -   a. Synthesis: As described in example 1-4        -   b. Modification: No further modifications        -   c. Purification: As described in example 1-4    -   4. Label preparation: In this example Labels were synthetic        oligonucleotides modified with biotin for coupling to the        Linker. 110 different Labels from the 1OS system were applied        (table 8).        -   a. Synthesis: As described in example 2-3.            -   i. The Label system used in this example were named 1OS                and comprised of single stranded oligonucleotides which                were applied as a DNA barcodes.        -   b. Modification: As described in example 1-4.        -   c. Purification: As described in example 1-4.    -   5. Detection Molecules preparation: 110 different Detection        Molecules were generated, each with a different Binding Molecule        encoded by a unique Label. The Binding Molecules (pMHCs) and        Labels (1OS DNA-barcodes) were attached to the Linker        (dextran-streptavidin-PE conjugate) to form Detection Molecules,        in such a way that a given pMHC was always attached to a given        DNA-barcode.        -   a. Detection Molecules were generated in the same way as            described in example 3. The given combination of Label            (DNA-barcode) and Binding Molecule (pMHC) of each Detection            Molecule are presented in table 9.        -   b. Modification: Since the total volume of 110 pooled            Detection Molecules exceeded 100 μl this volume was reduced            to reach a desired concentration of specific Binding            Molecules. This was done as described in example 2-4.        -   c. Purification: As described in example 1-4.    -   6. Incubation of sample and Detection Molecules: The cell sample        and the Detection Molecules were incubated in the same way as        described in example 1-2.    -   7. Enrichment of MHC molecules with desired characteristics: The        Detection Molecules were enriched for in the same way as        described in example 1-6.    -   8. Identification of enriched Detection Molecules: Because the        Detection Molecules were enriched based on specific interaction        of the Binding Molecule (the pMHC) with cells, the        identification of the associated Labels (DNA-barcodes) amongst        the sorted cells would also reveal the pMHCs that had bound to        cells in the PBMC sample. In this example the DNA-barcodes        associated with the enriched Detection Molecules, were amplified        by PCR and identified by high-throughput sequencing. The        enriched Labels, of the 1OS DNA-barcode system, were identified        as in example 3.

Example 5—Results and Conclusions

FACS sorting of fluorescent labeled cells, specific amplification ofDNA-barcode Labels and high-throughput sequencing verified that it waspossible to enrich and detect 1OS barcodes from a library of multipledifferent Detection molecules composed of 110 different 1OS DNA-barcodeLabels encoding for 110 different antigen specificities distributed on 6different HLA-types (FIG. 14 ). It was verified that severalDNA-barcodes, encoding different antigen specificities on differentHLA-types, could be enriched for and detected in parallel, indicativefor the presence of multiple antigen-specific T cell responses in thatsample.

This example demonstrates that it is possible to detect severalantigen-specific T cell responses in parallel when applying a library ofDetection molecules of increasing complexity.

Example 6

In this example 110 different binding molecules are used, the linker isdextran for all of the detection molecules and 110 different labels areused.

Example 6 Explained

This example was essentially the same as example 5 only another Labelsystem was applied.

This is an example where the Samples (1) were blood from six differentdonors with different HLA-types which were modified (1b) to generatePeripheral blood mononuclear cells (PBMCs).

The Linker (2) was a dextran conjugate with streptavidin andfluorochrome (Dextramer backbone from Immudex).

The Binding Molecules (3) were peptide-MHC (pMHC) complexes displayingone out of 110 different peptide-antigens comprised within 6 differentHLA-types. The MHC molecules were modified (3b) by biotinylation toprovide a biotin capture-tag for the Linker. The binding molecules werepurified (2c) by HPLC and quality controlled in terms of the formationof functional pMHC multimers for staining of control T cell populations.

The Labels (4) were oligonucleotides applied as DNA-barcodes. Theoligonucleotides were synthetized (4a) by DNA Technology A/S (Denmark)and were synthetically modified (4b) with a terminal biotin capture-tag.The labels were combined oligonucleotide labels arising by annealing anA oligonucleotide (modified with biotin) to a partially complimentary Boligonucleotide label followed by enzymatic DNA polymerase extension ofOligo A and Oligo B to create a fully double stranded label. TheDetection Molecule (5) was synthetized (5a) by attaching BindingMolecules in the form of biotinylated pMHC and Labels in the form ofbiotin-modified oligonucleotides (DNA-barcodes) onto astreptavidin-modified dextran linker. The detection molecule furthercontained a modification (5b) in the form of a fluorochrome. A libraryof 110 different Detection Molecules were generated wherein individualBinding Molecules, comprised of different pMHC, were encoded for bycorresponding individual Labels, comprised of different DNA-barcodes.

An amount of sample, PBMC's (1b) was incubated with an amount of mixedDetection Molecules (5) under conditions (6c) allowing binding ofDetection Molecules to T cells in the sample.

The cell-bound Detection Molecules were separated from the non-cellbound Detection Molecules (7) by first a few rounds of washing thePBMC's through centrifugation sedimentation of cells and resuspension inwash buffer followed by Fluorescence Activated Cell Sorting (FACS) offluorochrome labeled cells. T cells that can efficiently bind DetectionMolecules will fluoresce because of the fluorochrome comprised withinthe detection molecules; T cells that cannot bind detection moleculeswill not fluoresce. FACS-sorting leads to enrichment of fluorescentcells, and hence, enrichment of the detection molecules along with theassociated labels that bind T cells of the PBMC sample.

FACS isolated cells were subjected to PCR for specific amplification ofthe DNA-barcode associated with the Detection Molecules bound to theisolated cells. High throughput sequencing of the resultant PCR productrevealed the identity of Detection Molecules that bound to T cellspresent in the sample.

This example thus revealed the presence of T cells in the bloodexpressing a T cell receptor that binds to pMHC molecules representedwithin the library of Detection Molecules. The application of multiple(110) Detection Molecules in one sample enabled detection of T cellswith different T cell receptor specificities in parallel.

Example 6 in Detail

-   -   1. Sample preparation. The cell samples used in this example        were obtained by preparing PBMC's from blood drawn from six        different donors with a number of different peptide-antigen        responsive T cells, as determined by conventional pMHC-multimer.        -   a. Acquiring sample: Blood was obtained from the Danish            Blood Bank.        -   b. Modifying sample: PBMCs were isolated from whole blood as            described in example 1-2, 5.    -   2. Linker preparation: The linker used in this example was        prepared as in example 1-5.    -   3. Binding Molecules preparation: The Binding Molecules used in        this example were class I MHC-peptide complexes. The individual        specificities (allele and peptide combination) were generated as        described in example 1-5. A library of 110 different pMHCs,        comprised of 6 different HLA-types, were generated, these are        listed in table 9.        -   a. Synthesis: As described in example 1-5.        -   b. Modification: No further modifications        -   c. Purification: As described in example 1-5    -   4. Label preparation: In this example Labels were synthetic        oligonucleotides modified with biotin for coupling to the        Linker. 110 different Labels from the 2OS system were applied        (table 2).        -   a. Synthesis: As described in example 1, 4.            -   i. The Label system used in this example was named 2OS                and was developed to increase the complexity of a                limited number of oligonucleotide sequences. This was                enabled by applying a combinatorial strategy where two                partially complementary oligonucleotides (an A                oligonucleotide with a 5′ biotin tag and a B                oligonucleotide) where annealed and then elongated to                produce new unique oligonucleotide-sequences (AxBy)                which were applied as a molecular barcodes (Labels)                (FIG. 9 ). By combining 6 unique                oligonucleotide-sequences (A label precursor) that were                all partly complementary to 20 other unique                oligonucleotide sequences (B label precursor) a                combinatorial library of 120 different (AxBy) Labels                were produced. Only 110 of these Labels were used in                this example (table 9).        -   b. Modification: As described in example 1-5.        -   c. Purification: As described in example 1-5.    -   5. Detection Molecules preparation: 110 different Detection        Molecules were generated, each with a different Binding Molecule        encoded by a unique Label. The Binding Molecules (pMHCs) and        Labels (2OS DNA-barcodes) were attached to the Linker        (dextran-streptavidin-PE conjugate) to form Detection Molecules,        in such a way that a given pMHC was always attached to a given        DNA-barcode.        -   a. Detection Molecules were generated in the same way as            described in example 3, 4, 5. The given combination of Label            (2OS DNA-barcode) and Binding Molecule (pMHC) of each            Detection Molecule are presented in table 9.        -   b. Modification: Since the total volume of 110 pooled            Detection Molecules exceeded 100 μl this volume was reduced            to reach a desired concentration of specific Binding            Molecules (done as described in example 2-5).        -   c. Purification: As described in example 1-5.    -   6. Incubation of sample and Detection Molecules: The cell sample        and the Detection Molecules were incubated in the same way as        described in example 1-2, 5.    -   7. Enrichment of MHC molecules with desired characteristics: The        Detection Molecules were enriched for in the same way as        described in example 1-5.    -   8. Identification of enriched Detection Molecules: Because the        Detection Molecules were enriched based on specific binding of        the Binding Molecule (the pMHC) to cells, the identification of        the associated Labels (DNA-barcodes) amongst the sorted cells        would also reveal the pMHCs that had bound to cells in the PBMC        sample. In this example the DNA-barcodes associated with the        enriched Detection Molecules, were amplified by PCR and        identified by high-throughput sequencing. The enriched Labels,        of the 2OS DNA-barcode system, were identified as in example 4.

Example 6—Results and Conclusions

FACS sorting of fluorescent labeled cells, specific amplification ofDNA-barcode Labels and high-throughput sequencing verified that it waspossible to enrich and detect 2OS barcodes from a library of multipledifferent Detection molecules composed of 110 different 2OS DNA-barcodeLabels encoding for 110 different antigen specificities distributed on 6different HLA-types (FIG. 15 ). It was verified that severalDNA-barcodes, encoding different antigen specificities of differentHLA-types, could be enriched for and detected in parallel, indicativefor the presence of multiple antigen-specific T cell responses in thatsample.

This example demonstrates that it is possible to detect severalantigen-specific T cell responses in parallel when applying a library ofDetection molecules of increasing complexity.

Example 7

In this example 175 different pMHC complexes are used as bindingmolecules. The sample is tumor infiltrating Lymphocytes from a resectedtumor lesion of a human being.

Example 7 Explained

This is an example where the Samples (1) were resected tumor lesionsfrom 11 HLA-A0201 positive patients with malignant melanoma. The sampleswere modified (1b) to generate Tumor Infiltrating Lymphocytes (TILs).

The Linker (2) was a dextran conjugate with streptavidin andfluorochrome (Dextramer backbone from Immudex).

The Binding Molecules (3) were peptide-MHC (pMHC) complexes displayingone out of 175 different peptide-antigens. The MHC molecules weremodified (3b) by biotinylation to provide a biotin capture-tag for theLinker. The binding molecules were purified (2c) by HPLC and qualitycontrolled in terms of the formation of functional pMHC multimers forstaining of control T-cell populations.

The Labels (4) were oligonucleotides applied as DNA-barcodes. Theoligonucleotides were synthetized (4a) by DNA Technology A/S (Denmark)and were synthetically modified (4b) with a terminal biotin capture-tag.The labels were combined oligonucleotide labels arising by annealing anA oligonucleotide (modified with biotin) to a partially complimentary Boligonucleotide label followed by enzymatic DNA polymerase extension ofOligo A and Oligo B to create a fully double stranded label. TheDetection Molecule (5) was synthetized (5a) by attaching BindingMolecules in the form of biotinylated pMHC and Labels in the form ofbiotin-modified oligonucleotides (DNA-barcodes) onto astreptavidin-modified dextran linker. The detection molecule furthercontained a modification (5b) in the form of a fluorochrome. A libraryof 175 different Detection Molecules were generated wherein individualBinding Molecules, comprised of different pMHC, were encoded for bycorresponding individual Labels, comprised of different DNA-barcodes.

An amount of sample, TILs (1b), was incubated with an amount of mixedDetection Molecules (5) under conditions (6c) allowing binding ofDetection Molecules to T cells in the sample.

The cell-bound Detection Molecules were separated from the non-cellbound Detection Molecules (7) by first a few rounds of washing the TIL'sthrough centrifugation sedimentation of cells and resuspension in washbuffer followed by Fluorescence Activated Cell Sorting (FACS) offluorochrome labeled cells. T cells that can efficiently bind DetectionMolecules will fluoresce because of the fluorochrome comprised withinthe detection molecules; T cells that cannot bind detection moleculeswill not fluoresce. FACS-sorting leads to enrichment of fluorescentcells, and hence, enrichment of the detection molecules along with theassociated labels that bind T cells of the TIL sample.

FACS isolated cells were subjected to PCR for specific amplification ofthe DNA-barcode Label associated with the Detection Molecules bound tothe isolated cells. High throughput sequencing of the resultant PCRproduct revealed the identity of Detection Molecules that bound to Tcells present in the sample.

This example thus revealed the presence of T cells among the TIL'sexpressing a T cell receptor that binds to pMHC molecules representedwithin the library of Detection Molecules. The number of sequencingreads mapped to a given DNA-barcode and its corresponding BindingMolecule would mirror the frequency of the T cells found by conventionalMHC multimer stainings using the same Binding Molecules. The applicationof multiple (175) Detection Molecules in one sample enabled detection ofT cells with different T cell receptor specificities in parallel. Thisis feasible also when the sample, as in present example is TILs that onaverage basis has a lower avidity of their TCR to their pMHC antigen.Consequently they are more challenging to detect than virus-responsive Tcells using traditional fluorescence labelled MHC mulitimers.

Example 7 in Detail

-   -   1. Sample preparation. The 11 cell samples used in this example        were obtained from resected tumor lesions obtained from patients        with malignant melanoma.        -   a. Acquiring sample. TILs were derived from tumor fragments            from melanoma patients. Tumor lesion were resected following            surgical removal of the given tumor lesion.        -   b. Modifying sample. Tumor fragments (1-3 mm3) were cultured            individually in complete medium (RPMI with 10% human serum,            100 U/ml penicillin, 100 μg/ml streptomycin, 1.25 μg/ml            fungizone [Bristol-Myers Squibb] and 6000 U/ml IL-2) at            37° C. and 5% CO2, allowing TILs to migrate to the medium.            TILs were expanded to reach >50×106 total cells originated            from approximately 24 fragments, which had expanded to            confluent growth in 2-ml wells and eliminated adherent tumor            cells (average of approximately 2×106 cells per well from            each tumor fragment). TIL cultures were expanded on a            clinical scale using a standard rapid expansion protocol            (REP). Briefly, TILs were stimulated with 30 ng/ml anti-CD3            antibody (OKT-3; Ortho Biotech) and 6000 U/ml IL-2 in the            presence of irradiated (40 Gy) allogenic feeder cells (PBMCs            from a healthy donor) at a feeder:TIL ratio of 200:1.            Initially, TILs were rapidly expanded in a 1:1 mix of            complete medium and REP medium (AIM-V [Invitrogen] with 10%            human serum, 1.25 μg/ml fungizone and 6000 U/ml IL-2), but            after seven days complete medium and serum were removed            stepwise from the culture by adding REP medium without serum            to maintain cell densities around 1-2×106 cells/ml. TIL            cultures were cryopreserved at −150° C. in human serum            containing 10% DMSO. All patients were HLA-A0201 positive.    -   2. Linker preparation: The linker used in this example was        prepared as in example 1-6.    -   3. Binding Molecules preparation: The Binding Molecules used in        this example were class I MHC-peptide complexes        (peptide-HLA-A0201). The individual specificities (allele and        peptide combination) were generated as described in example 1-6.        A library of 175 different pMHCs were generated, these are        listed in table 2.        -   a. Synthesis: As described in example 1-6        -   b. Modification: No further modifications        -   c. Purification: As described in example 1-6    -   4. Label preparation: In this example Labels were synthetic        oligonucleotides modified with biotin for coupling to the        Linker. 175 different Labels from the 2OS system were applied        (table 14).        -   a. Synthesis: As described in example 1.            -   i. The Label system used in this example was named 2OS                and was developed to increase the complexity of a                limited number of oligonucleotide sequences. This was                enabled by applying a combinatorial strategy where two                partially complementary oligonucleotides (an A                oligonucleotide with a 5′ biotin tag and a B                oligonucleotide) where annealed and then elongated to                produce new unique oligonucleotide-sequences (AxBy)                which were applied as a DNA-barcodes (Labels) (FIG. 9 ).                By combining 22 unique oligonucleotide-sequences (A                label precursor) that are all partly complementary to 55                other unique oligonucleotide sequences (B label                precursor) a combinatorial library of 1,210 different                (AxBy) Labels were produced. Only 175 of these Labels                were used in this example (table 14). Refer to table 2                for an overview of the different 2OS A and B nucleotide                sequences.        -   b. Modification: As described in example 1-6.        -   c. Purification: As described in example 1-6.    -   5. Detection Molecules preparation: 175 different Detection        Molecules were generated, each with a different Binding Molecule        encoded by a unique Label. The Binding Molecules (pMHCs) and        Labels (2OS DNA-barcodes) were attached to the Linker        (dextran-streptavidin-PE conjugate), to form Detection        Molecules, in such a way that a given pMHC was always attached        to a given DNA-barcode.        -   a. Detection Molecules were essentially generated in the            same way as described in example 1-6. The given combination            of Label (2OS DNA-barcode) and Binding Molecule (pMHC) of            each Detection Molecule are presented in table 14.        -   b. Modification: Since the total volume of 175 pooled            Detection Molecules exceeded 100 μl this volume was reduced            to reach a desired concentration of specific Binding            Molecules. This was done as described in example 2-6.        -   c. Purification: As described in example 1-6.    -   6. Incubation of sample and Detection Molecules: The cell sample        and the Detection Molecules were mixed in one container to allow        Detection Molecules to bind to T cells.        -   a. Amount of sample: Between 0.2×10E6-2.3×10E6 cells in the            form of TILs, were used.        -   b. Amount of Detection Molecule: As described in example 1-6        -   c. Conditions: Samples and Detection Molecules were treated            as described in example 1-2 and 5-6.    -   7. Enrichment of MHC molecules with desired characteristics: The        Detection Molecules were enriched for in the same way as        described in example 1-6.    -   8. Identification of enriched Detection Molecules: Because the        Detection Molecules were enriched based on specific interaction        of the Binding Molecule (the pMHC) with cells, the        identification of the associated Labels (DNA-barcodes) amongst        the sorted cells would also reveal the pMHCs that had bound to        cells in the TIL sample. In this example the DNA-barcodes        associated with the enriched Detection Molecules, were amplified        by PCR and identified by high-throughput sequencing.        -   a. Apply. The sorted cell samples which contained            DNA-barcodes derived from the enriched Detection Molecules            were amplified by PCR. See table 10 for composition of the            PCR and table 11 for the thermal profile. The Taq PCR Master            Mix Kit (Qiagen, #201443) was applied and PCR was run on the            thermal cycler: GeneAmp, PCR System 9700 (Applied            Biosystem). PCR products were visualized after gel            electrophoresis on a Bio-Rad Gel Doc EZ Imager. DNA was            sequenced using the Ion Torrent PGM platform (Life            Technologies)            -   i. Primers were purchased from DNA Technology (Denmark)                and delivered as lyophilized powder. Stock dilutions of                100 μM were made in nuclease free water and stored at                −20° C. The primers included adaptors for Ion Torrent                sequencing, i.e. an A-key and a P1-key on the forward                and reverse primer respectively. Additionally the                forward primers had unique DNA sequences besides the                primer region and the A-key. These primers were used to                assign DNA-barcodes derived from the same sample with a                sample-identification sequence (Sample-ID barcode)                (refer to table 13 for primer sequences). This enabled                distribution of DNA-barcode sequence reads according to                their originating sample, when DNA-barcodes from                multiple samples were sequenced in the same sequencing                reaction (see FIG. 9 for a schematic presentation of the                primer design). If >10.000 cells were sorted in the                enrichment step, only a volume corresponding to 10.000                cells were applied as template in the PCR. To further                examine the potential impact on the number of cells                selected with associated Detection molecules, we                included Enriched Detection Molecules derived from one                sample (sample 8) multiple times, so that triplicate                PCRs were run with equivalent to 10.000 sorted cells and                100 sorted cells respectively. Moreover a single PCR was                run with 1000 cells from the same sample. The                non-enriched library of the 175 different Detection                Molecules (diluted 10.000x after being reduced in                volume) were also assigned with a sample-ID barcode                through PCR (referred to as the Detection                Molecule-input). Triplicate PCRs were run with the                Detection Molecule-input. Information about the                distribution of Labels within the library of Detection                Molecules before enrichment would allow normalization of                the sequence output. Pooled PCR products derived from                the Detection Molecule-input and from multiple                incubations of Detection Molecule and sample were                separated according to size, by gel-electrophoresis, and                the excised gel-fragment containing DNA-fragments of                ˜200 bp were purified with the QIAquick PCR Purification                Kit (Qiagen, #28104) according to standard procedure.            -   ii. The purified DNA was sequenced by Amplexa (Denmark)                on an Ion Torrent PGM 314 chip.        -   b. Analysis. Positive sequence reads were aligned to            sequences that read from the sample-barcode-identity at the            5′-end all the way through the DNA-barcode-identity. The            number of reads was normalized according to the total number            of reads that mapped to the same sample-ID barcode and            according to the Detection Molecule input reads. The            Analysis was essentially as in example 4 and 6 except that            the database of 2OS sequences was expanded to include the            new 175 Labels and sequences were mapped according to            corresponding 2OS DNA-barcodes.            -   i. Mapping sequencing reads to 2OS DNA-barcodes: A                sequence database was created consisting of the possible                combinations of 20 sample-identification barcodes and                192 2OS DNA barcodes (together with the primer and                annealing sequences from the 2OS system). This                accumulated to 3840 sequences that could be expected                from a sequencing run. Each sequencing read was then                used to search the database for alignments, using the                nucleotide BLAST algorithm, with a match reward of 1,                mismatch reward of −2 and a gap cost of 2 for both                opening and extending a gap. In this way sequencing                errors were penalized equally, whether a base was                miscalled or inserted/deleted in the sequencing read                compared to the actual sequence. Alignments were                discarded by the following criteria:                -   1. E-value >1e-12; insufficient length of alignment                    (should be greater than 102 for the 2OS barcodes).                -   2. Start position in subject sequence larger than 2,                    i.e. fewer than 5 out of 6 bases in the unique part                    of the sample-identification barcode was included in                    the alignment.            -   ii. If multiple alignments could still be found for any                sequencing read, only the alignment with the best                percent identity was kept. Finally, the number of reads                mapping to each DNA barcode in the database was counted.            -   iii. Identifying overrepresented DNA barcodes: Two                amendments of the analysis performed in example 4 and 6                (as well as 3 and 5 in respect to the 1OS label system)                were introduced:                -   a. The N6 sequence (FIG. 9 ) were applied to reduce                    the clonality of the amplification product to avoid                    potential biases derived from amplification of                    enriched 2OS sequence Labels.                -    i. Any repetitive reads that would share a N6 and                    map to a given sample-ID all through the 2OS barcode                    region were virtually removed ensuring that this                    sequence was only accounted for once.                -   b. The sequencing data was reanalyzed after virtual                    removal of the most high-frequent sequences. This                    ensured that potential biases derived from                    high-percentage enrichment of a given Detection                    Molecule, resulting in large read counts from the                    associated 2OS sequence Label, would not mask the                    presence of lower percentages yet enriched Detection                    Molecules.            -   Collectively this strategy is termed clonality                reduction. Relative read counts were calculated by                normalizing each read to the total read count mapping to                the same sample-ID barcode. The relative read counts                were then used to calculate the fold change per                DNA-barcode compared to the control DNA-barcode                Detection Molecule input (the non-enriched Detection                Molecule library). Significantly overrepresented                DNA-barcodes were identified using a 2-sample test for                equality of proportions on the raw read counts in a                sample versus the DNA-barcode input-sample, and p-values                were corrected for multiple testing using the                Benjamini-Hochberg FDR method. The algorithm applied for                this analysis is available at:                www.cbs.dtu.dk/services/Barracoda/

Example 7—Results and Conclusions

FACS sorting of fluorescent labeled cells, specific amplification ofDNA-barcode Labels and high-throughput sequencing verified that it waspossible to enrich and detect 2OS barcodes from a library of multipledifferent Detection molecules composed of 175 different 2OS DNA-barcodeLabels encoding for 175 different cancer-specificities (FIG. 16 ). Itimplied that several DNA-barcodes, encoding different antigenspecificities, could be enriched for and detected in parallel, also whenthe samples, as in the present example, were TILs that on average has alower avidity of their TCR to their pMHC antigen. The results indicatedthat the presence of multiple cancer-specific T cell responses could bedetected in parallel in a sample.

This example implies that it is possible to detect severalcancer-specific T cell responses in parallel when applying a library ofDetection molecules of increasing complexity.

Example 8

In this example 1025 different binding molecules are used in the form ofpMHC complexes (each with DNA labels).

The sample is a mixture of two different blood samples, the isolatingand/or detecting is done by flow cytometry and the determining of theidentity of the label is done by sequencing the DNA label.

Example 8 Explained

This example is essentially the same as example 4 but will apply alarger Detection Molecule library (1025) and will include a greaternumber of different HLA-types (11) to analyze the same samples.

This is an example where the Samples (1) are blood from one donor thatis HLA-B0702:CMV pp65 TPR positive and another donor that is HLA-B0702negative which are modified (1b) to generate Peripheral bloodmononuclear cells (PBMCs). These samples are mixed in different ratiosto generate new samples with different but known frequencies of T cellsspecific toward the HLA-B0702:CMV epitope.

The Linker (2) is a dextran conjugate with streptavidin and fluorochrome(Dextramer backbone from Immudex).

The Binding Molecules (3) are peptide-MHC (pMHC) complexes displayingone out of 1025 different peptide-antigens. The MHC molecules aremodified (3b) by biotinylation to provide a biotin capture-tag for theLinker. The binding molecules are purified (2c) by HPLC and qualitycontrolled in terms of the formation of functional pMHC multimers forstaining of control T-cell populations.

The Labels (4) are oligonucleotides applied as DNA-barcodes. Theoligonucleotides are synthetized (4a) by DNA Technology A/S (Denmark)and are synthetically modified (4b) with a terminal biotin capture-tag.The labels are combined oligonucleotide labels arising by annealing an Aoligonucleotide (modified with biotin) to a partially complimentary Boligonucleotide label followed by enzymatic DNA polymerase extension ofOligo A and Oligo B to create a fully double stranded label.

The Detection Molecule (5) is synthetized (5a) by attaching BindingMolecules in the form of biotinylated pMHC and Labels in the form ofbiotin-modified oligonucleotides (DNA-barcodes) onto astreptavidin-modified dextran linker. The detection molecule furthercontains a modification (5b) in the form of a fluorochrome. A library of1025 different Detection Molecules are generated wherein individualBinding Molecules, comprised of different pMHC, are encoded for bycorresponding individual Labels, comprised of different DNA-barcodes.

An amount of sample, PBMC's (1b) is incubated with an amount of mixedDetection Molecules (5) under conditions (6c) allowing binding ofDetection Molecules to T cells in the sample.

The cell-bound Detection Molecules are separated from the non-cell boundDetection Molecules (7) by first a few rounds of washing the PBMC'sthrough centrifugation sedimentation of cells and resuspension in washbuffer followed by Fluorescence Activated Cell Sorting (FACS) offluorochrome labeled cells. T cells that can efficiently bind DetectionMolecules will fluoresce because of the fluorochrome comprised withinthe detection molecules; T cells that cannot bind detection moleculeswill not fluoresce. FACS-sorting leads to enrichment of fluorescentcells, and hence, enrichment of the detection molecules along with theassociated labels that bind T cells of the PBMC sample.

FACS isolated cells are subjected to PCR for specific amplification ofthe DNA-barcode associated with the Detection Molecules bound to theisolated cells. High throughput sequencing of the resultant PCR productwill reveal the identity of Detection Molecules that binds to T cellspresent in the sample.

This example will reveal the presence of T cells in the blood expressinga T cell receptor that binds to pMHC molecules represented within thelibrary of 1025 Detection Molecules. The number of sequencing reads thatwill map to a given DNA-barcode and its corresponding Binding Moleculewill mirror the frequency of the T cells found by conventional MHCmultimer stainings using the same Binding Molecules. The increasedcomplexity of the Detection Molecule library, in terms of the number ofdifferent Binding Molecules and Labels, will reflect positively upon thesensitivity for detecting a given Label, i.e. a Detection Molecule,associated with low frequencies of T cells binding such DetectionMolecules.

Example 8 in Detail

-   -   1. Sample preparation. The cell samples used in this example are        obtained by preparing PBMC's from blood drawn from one donor        that is HLA-B0702:CMV pp65 TPR positive and from another donor        that is HLA-B0702 negative, as determined by conventional        pMHC-multimer and antibody staining. They are acquired (a.) and        modified (b.) in the same way as described in example 3-4.    -   2. Linker preparation: The linker used in this example is        prepared as in example 1-7.    -   3. Binding Molecules preparation: The Binding Molecules used in        this example are class I MHC-peptide complexes. The individual        specificities (allele and peptide combination) are generated as        described in example 1-7. A library of 1025 different pMHCs are        generated including 11 different HLA-types.        -   a. Synthesis: As described in example 1-7        -   b. Modification: No further modifications        -   c. Purification: As described in example 1-7    -   4. Label preparation: In this example Labels are synthetic        oligonucleotides modified with biotin for coupling to the        Linker. 1025 different Labels from the 2OS system were applied        (table 2).        -   a. Synthesis: As described in example 1.            -   i. The Label system used in this example is named 2OS                and was developed to increase the complexity of a                limited number of oligonucleotide sequences. This is                enabled by applying a combinatorial strategy where two                partially complementary oligonucleotides (an A                oligonucleotide with a 5′ biotin tag and a B                oligonucleotide) are annealed and then elongated to                produce new unique oligonucleotide-sequences (AxBy)                which are applied as a DNA-barcodes (Labels) (FIG. 2 ).                By combining 22 unique oligonucleotide-sequences (A                label precursor) that are all partly complementary to 55                other unique oligonucleotide sequences (B label                precursor) a combinatorial library of 1210 different                (AxBy) Labels are produced. Only 1025 of these Labels                are used in this example.        -   b. Modification: As described in example 1-7.        -   c. Purification: As described in example 1-7.    -   5. Detection Molecules preparation: 1025 different Detection        Molecules are generated, each with a different Binding Molecule        encoded by a unique Label. The Binding Molecules (pMHCs) and        Labels (2OS DNA-barcodes) are attached to the Linker        (dextran-streptavidin-PE conjugate), to form Detection        Molecules, in such a way that a given pMHC is always attached to        a given DNA-barcode.        -   a. Detection Molecules are essentially generated in the same            way as described in example 1-7. The given combination of            Label (2OS DNA-barcode) and Binding Molecule (pMHC) of each            Detection Molecule are registered.        -   b. Modification: Since the total volume of 1025 pooled            Detection Molecules exceeds 100 μl this volume is reduced to            reach a desired concentration of specific Binding Molecules.            This is done as described in example 2-7.        -   c. Purification: As described in example 1-7.    -   6. Incubation of sample and Detection Molecules: The cell sample        and the Detection Molecules are mixed in one container to allow        Detection Molecules to bind to T cells.        -   a. Amount of sample: Samples are equivalent to those used in            example 3-4.        -   b. Amount of Detection Molecule: As described in example 1-7        -   c. Conditions: Samples and Detection Molecules are treated            under the same conditions as described in example 3-4.    -   7. Enrichment of MHC molecules with desired characteristics: The        Detection Molecules are enriched for in the same way as        described in example 1-7.    -   8. Identification of enriched Detection Molecules: Because the        Detection Molecules are enriched based on specific interaction        of the Binding Molecule (the pMHC) with cells, the        identification of the associated Labels (DNA-barcodes) amongst        the sorted cells will also reveal the pMHCs that bind to cells        in the PBMC sample. In this example the DNA-barcodes associated        with the enriched Detection Molecules, are amplified by PCR and        identified by high-throughput sequencing.        -   a. Apply. The sorted cell samples which contain DNA-barcodes            derived from the enriched Detection Molecules are amplified            by PCR. See table 10 for composition of the PCR and table 11            for the thermal profile. The Taq PCR Master Mix Kit (Qiagen,            #201443) is applied and PCR is run on the thermal cycler:            GeneAmp, PCR System 9700 (Applied Biosystem). PCR products            are visualized after gel electrophoresis on a Bio-Rad Gel            Doc EZ Imager. DNA is sequenced using the Ion Torrent PGM            platform (Life Technologies)            -   i. Primers are purchased from DNA Technology (Denmark)                and delivered as lyophilized powder. Stock dilutions of                100 μM are made in nuclease free water and stored at                −20° C. The primers include adaptors for Ion Torrent                sequencing, i.e. an A-key and a P1-key on the forward                and reverse primer respectively. Additionally the                forward primers have unique DNA sequences besides the                primer region and the A-key. These primers are used to                assign DNA-barcodes derived from the same sample with a                sample-identification sequence (Sample-ID barcode)                (refer to table 13 for primer sequences). This enables                distribution of DNA-barcode sequence reads according to                their originating sample, when DNA-barcodes from                multiple samples are sequenced in the same sequencing                reaction (see FIG. 9 for a schematic presentation of the                primer design). The non-enriched library of the 1025                different Detection Molecules (diluted 100.000× after                being reduced in volume) are also assigned with a                sample-ID barcode through PCR (referred to as the                Detection Molecule input). Triplicate PCRs are run with                the Detection Molecule-input. Information about the                distribution of Labels within the library of Detection                Molecules before enrichment will allow normalization of                the sequence output. Pooled PCR products derived from                the sample input and from multiple incubations of                Detection Molecule and sample are separated according to                size, by gel-electrophoresis, and the excised                gel-fragment containing DNA-fragments of ˜200 bp are                purified with the QIAquick PCR Purification Kit (Qiagen,                #28104) according to standard procedure.            -   ii. The purified DNA was sequenced on an Ion Torrent PGM                314 chip.        -   b. Analysis. Positive sequence reads are aligned to            sequences that read from the sample-barcode-identity at the            5′-end all the way through the DNA-barcode-identity. The            number of reads is normalized according to the total number            of reads that maps to the same sample-ID barcode and            according to the Detection Molecule input reads. The            Analysis is essentially as in example 7 except that the            database of 2OS sequences now includes 1025 Labels and            sequences are mapped according to corresponding 2OS            DNA-barcodes.            -   i. Mapping sequencing reads to 2OS DNA-barcodes: A                sequence database is created consisting of the possible                combinations of 10 sample-identification barcodes and                1025 2OS DNA barcodes (together with the primer and                annealing sequences from the 2OS system). This                accumulates to 10250 sequences that can be expected from                a sequencing run. Each sequencing read is then used to                search the database for alignments, using the nucleotide                BLAST algorithm, with a match reward of 1, mismatch                reward of −2 and a gap cost of 2 for both opening and                extending a gap. In this way sequencing errors are                penalized equally, whether a base was miscalled or                inserted/deleted in the sequencing read compared to the                actual sequence. Alignments are discarded by the                following criteria:                -   1. E-value >1e-12; insufficient length of alignment                    (should be greater than 102 for the 2OS barcodes).                -   2. Start position in subject sequence larger than 2,                    i.e. fewer than 5 out of 6 bases in the unique part                    of the sample-identification barcode was included in                    the alignment.            -   If multiple alignments can still be found for any                sequencing read, only the alignment with the best                percent identity is kept. Finally, the number of reads                mapping to each DNA barcode in the database is counted.            -   ii. Identifying overrepresented DNA barcodes: Is                essentially performed as described in example 7 with the                strategy of applying clonality reduction.                -   Relative read counts are calculated by normalizing                    each read to the total read count mapping to the                    same sample-ID barcode. The relative read counts are                    then used to calculate the fold change per                    DNA-barcode compared to the control DNA-barcode                    Detection Molecule input (the non-enriched Detection                    Molecule library). Significantly overrepresented                    DNA-barcodes are identified using a 2-sample test                    for equality of proportions on the raw read counts                    in a sample versus the DNA-barcode input-sample, and                    p-values are corrected for multiple testing using                    the Benjamini-Hochberg FDR method. The algorithm                    applied for this analysis is available at:                    www.cbs.dtu.dk/services/Barracoda/

Example 8—Results and Conclusions

The expected outcome of this example is knowledge on the sensitivity fordetecting antigen-specific T cell responses of decreasing frequency(<0.002% of CD8 T cells) in a number of similar samples. The sensitivitywill expectantly increase with increasing numbers of Detection moleculesincubated with a given sample, because any sequencing reads that arecaused by background will be distributed on a greater number of Labels,in this example 1025.

Example 9

In this example 1025 different pMHC complexes, comprising 11 differentHLA-alleles, are used as binding molecules. The sample is a mixture ofblood from 10 different donors.

Example 9 Explained

This example is essentially the same as example 6 but a greater numberof samples (10) will be analyzed with a larger Detection Moleculelibrary (1025), which will include a greater number of differentHLA-types (11).

This is an example where the Samples (1) are blood from 10 differentdonors with different HLA-types which are modified (1b) to generatePeripheral blood mononuclear cells (PBMCs).

The Linker (2) is a dextran conjugate with streptavidin and fluorochrome(Dextramer backbone from Immudex).

The Binding Molecules (3) are peptide-MHC (pMHC) complexes displayingone out of 1025 different peptide-antigens. The MHC molecules aremodified (3b) by biotinylation to provide a biotin capture-tag for theLinker. The binding molecules are purified (2c) by HPLC and qualitycontrolled in terms of the formation of functional pMHC multimers forstaining of control T-cell populations.

The Labels (4) are oligonucleotides applied as DNA-barcodes. Theoligonucleotides are synthetized (4a) by DNA Technology A/S (Denmark)and are synthetically modified (4b) with a terminal biotin capture-tag.The labels are combined oligonucleotide labels arising by annealing an Aoligonucleotide (modified with biotin) to a partially complimentary Boligonucleotide label followed by enzymatic DNA polymerase extension ofOligo A and Oligo B to create a fully double stranded label.

The Detection Molecule (5) is synthetized (5a) by attaching BindingMolecules in the form of biotinylated pMHC and Labels in the form ofbiotin-modified oligonucleotides (DNA-barcodes) onto astreptavidin-modified dextran linker. The detection molecule furthercontains a modification (5b) in the form of a fluorochrome. A library of1025 different Detection Molecules are generated wherein individualBinding Molecules, comprised of different pMHC, are encoded for bycorresponding individual Labels, comprised of different DNA-barcodes.

An amount of sample, PBMC's (1b) is incubated with an amount of mixedDetection Molecules (5) under conditions (6c) allowing binding ofDetection Molecules to T cells in the sample.

The cell-bound Detection Molecules are separated from the non-cell boundDetection Molecules (7) by first a few rounds of washing the PBMC'sthrough centrifugation sedimentation of cells and resuspension in washbuffer followed by Fluorescence Activated Cell Sorting (FACS) offluorochrome labeled cells. T cells that can efficiently bind DetectionMolecules will fluoresce because of the fluorochrome comprised withinthe detection molecules; T cells that cannot bind detection moleculeswill not fluoresce. FACS-sorting leads to enrichment of fluorescentcells, and hence, enrichment of the detection molecules along with theassociated labels that bind T cells of the PBMC sample.

FACS isolated cells are subjected to PCR for specific amplification ofthe DNA-barcode associated with the Detection Molecules bound to theisolated cells. High throughput sequencing of the resultant PCR productwill reveal the identity of Detection Molecules that binds to T cellspresent in the sample.

This example will reveal the presence of T cells in the blood expressinga T cell receptor that binds to pMHC molecules represented within thelibrary of 1025 Detection Molecules. The number of sequencing reads thatwill map to a given DNA-barcode and its corresponding Binding Moleculewill mirror the frequency of the T cells found by conventional MHCmultimer stainings using the same Binding Molecules. The increasedcomplexity of the Detection Molecule library, in terms of the number ofdifferent Binding Molecules and Labels, will enable detection of T cellsof 1025 different T cell receptor specificities in parallel and willreflect positively upon the sensitivity for detecting a given Label,i.e. a Detection Molecule, associated with low frequencies of T cellsbinding such Detection Molecules.

Example 9 in Detail

-   -   1. Sample preparation. The cell samples used in this example are        obtained by preparing PBMC's from blood drawn from 10 different        donors with a number of different peptide-antigen responsive T        cells, as determined by conventional pMHC-multimer.        -   a. Acquiring sample: Blood is obtained from the Danish Blood            Bank.        -   b. Modifying sample: PBMCs are isolated from whole blood as            described in example 1-2, 5-6.    -   2. Linker preparation: The linker used in this example is        prepared as in example 1-7 and example 8.    -   3. Binding Molecules preparation: The Binding Molecules used in        this example are class I MHC-peptide complexes. The individual        specificities (allele and peptide combination) are generated as        described in example 1-7 and example 8. A library of 1025        different pMHCs are generated including 10 different HLA-types.        -   a. Synthesis: As described in example 1-7 and example 8.        -   b. Modification: No further modifications        -   c. Purification: As described in example 1-7 and example 8.    -   4. Label preparation: In this example Labels are synthetic        oligonucleotides modified with biotin for coupling to the        Linker. 1025 different Labels from the 2OS system were applied        (table 2).        -   a. Synthesis: As described in example 1.            -   i. The Label system used in this example is named 2OS                and was developed to increase the complexity of a                limited number of oligonucleotide sequences. This is                enabled by applying a combinatorial strategy where two                partially complementary oligonucleotides (an A                oligonucleotide with a 5′ biotin tag and a B                oligonucleotide) are annealed and then elongated to                produce new unique oligonucleotide-sequences (AxBy)                which are applied as a DNA-barcodes (Labels) (FIG. 9 ).                By combining 22 unique oligonucleotide-sequences (A                label precursor) that are all partly complementary to 55                other unique oligonucleotide sequences (B label                precursor) a combinatorial library of 1210 different                (AxBy) Labels are produced. Only 1025 of these Labels                are used in this example.        -   b. Modification: As described in example 1-7 and example 8.        -   c. Purification: As described in example 1-7 and example 8.    -   5. Detection Molecules preparation: 1025 different Detection        Molecules are generated, each with a different Binding Molecule        encoded by a unique Label. The Binding Molecules (pMHCs) and        Labels (2OS DNA-barcodes) are attached to the Linker        (dextran-streptavidin-PE conjugate), to form Detection        Molecules, in such a way that a given pMHC is always attached to        a given DNA-barcode.        -   a. Detection Molecules are essentially generated in the same            way as described in example 1-7 and example 8. The given            combination of Label (2OS DNA-barcode) and Binding Molecule            (pMHC) of each Detection Molecule are registered.        -   b. Modification: Since the total volume of 1025 pooled            Detection Molecules exceeds 100 μl this volume is reduced to            reach a desired concentration of specific Binding Molecules.            This is done as described in example 2-7 and example 8.        -   c. Purification: As described in example 1-7 and example 8.    -   6. Incubation of sample and Detection Molecules: The cell sample        and the Detection Molecules are incubated in the same way as        described in example 1-2, 5-7. The cell sample and the Detection        Molecules are mixed in one container to allow Detection        Molecules to bind to T cells.    -   7. Enrichment of MHC molecules with desired characteristics: The        Detection Molecules are enriched for in the same way as        described in example 1-7 and example 8.    -   8. Identification of enriched Detection Molecules: Because the        Detection Molecules are enriched based on specific interaction        of the Binding Molecule (the pMHC) with cells, the        identification of the associated Labels (DNA-barcodes) amongst        the sorted cells will also reveal the pMHCs that bind to cells        in the PBMC sample. In this example the DNA-barcodes associated        with the enriched Detection Molecules, are amplified by PCR and        identified by high-throughput sequencing. The enriched Labels,        of the 2OS DNA-barcode system, are identified as in example 8.

Example 9—Results and Conclusions

The expected outcome of this example is knowledge of the potentialcomplexity for detecting multiple antigen-specific T cell responses inparallel in a single sample. Since the sensitivity is expected toincrease with increasing numbers of Detection molecules incubated with agiven sample (as described in example 8) it is expected that multiplemore T cell responses will also be detected in parallel when using aDetection molecule library of the said complexity. The example will thusprove that it is possible to detect multiple antigen-specific T cellresponses in parallel when applying a Detection molecule librarycomprised of 1025 different 2OS Labels encoding 1025 different Bindingmolecules distributed on 11 HLA-types.

Example 10

In this example it is shown how multiple, single-cell analyses can berapidly performed using the present invention. The isolating and/ordetecting is done by FACS, leading to the identification of a T cell andits TCR, as well as the pMHC complex that recognizes the T cell bybinding to the TCR.

Example 10 Explained

This example will describe how the detection of Binding Molecules can belinked to the T cell receptor (TCR) sequence following single cellsorting of T-cells associated with Detection Molecules.

This is an example where the Sample (1) will be a mixture of T cellscomprising TCRs of interest. This could e.g. be tumor infiltratinglymphocytes from a cancer patient. The Linker (2) is a dextran conjugatewith streptavidin and fluorochrome (Dextramer backbone from Immudex).

The Binding Molecules (3) is peptide-MHC (pMHC) complexes displaying aof library peptides that are potentially recognized by the T cells inthe Sample. This could be a library of melanoma-associated peptides, asused in example 7, or it could be a library of personally-definedpotential epitope sequences based on the characteristics of the givenpatient's tumor. This could e.g. be mutation-derived T cell epitopesand/or epitopes selected based on the expression pattern in theindividual tumor. The Binding Molecules will be modified (3b) bybiotinylation and quality controlled as described in example 1-6.

The Labels (4) are oligonucleotides as in example 7-9.

The detection molecule (5) will be synthetized (5a) and modified (5b) asin example 7-9.

An amount of Sample, Tumor infiltrating lymphocytes (1b) will be mixedwith detection molecules (5) under conditions (6c) that allow binding ofdetection molecules to T cells in the sample.

Cells will be FACS sorted based on the attachment of DetectionMolecules, transferred to a FLUIDIGM C1 unit (or similar device) forsingle-cell amplification of nucleotide labels and T cell receptorgenes.

This example thus reveals the possibility to identify, on a single-celllevel, both the antigen-specificity and the T cell receptor sequence.This being done in a mixture of multiple different Binding Molecules(potentially, but not exclusively, >1000). This technology provides amean for high-throughput identification of TCRs combined with adescription of the antigen specificity of the TCR.

Example 10 in Detail

-   -   1. Sample preparation. The cell sample to be used in this        example is a collection of T cells with a recognition profile of        interest for determining the sequences of the TCRs associated        with this recognition. The sample could e.g. be tumor        infiltration lymphocytes from a cancer patient. As described in        example 7.        -   c. Acquiring sample: As in example 7.        -   d. Modifying sample: As in example 7.    -   2. Linker preparation: The linker used in this example is        prepared as in example 1-10. The fluorochrome co-attachment is        important for the selection process in the example.    -   3. Binding molecule preparation: The binding molecules used in        this example will be a collection of peptide-MHC molecules,        designed to match the T cell reactivity in the sample. This        could be a library of melanoma-associated peptides, as used in        example 7, or it could be a library of personally-defined        potential epitope sequences based on the characteristics of the        given patient's tumor. This could e.g. be mutation-derived T        cell epitopes and/or epitopes selected based on the expression        pattern if the individual tumor.        -   a. Synthesis: as in example 7-9.        -   b. Modification: No further modifications        -   c. Purification: as in example 7-9.    -   4. Label preparation: as in example 7-9.    -   5. Detection Molecules preparation: multiple (<1000) different        Detection Molecules are generated, each with a different Binding        Molecule encoded by a unique Label. The Binding Molecules        (pMHCs) and Labels (2OS DNA-barcodes) are attached to the Linker        (dextran-streptavidin-PE conjugate), to form Detection        Molecules, in such a way that a given pMHC is always attached to        a given DNA-barcode.        -   a. Detection Molecules are essentially generated in the same            way as described in example 1-8.        -   b. Modification: Since the total volume of >1000 pooled            Detection Molecules exceeds 100 μl this volume is reduced to            reach a desired concentration of specific Binding Molecules.            This is done as described in example 2-8.        -   c. Purification: As described in example 1-8.    -   6. Incubation of sample and Detection Molecules: The cell sample        and the Detection Molecules are incubated in the same way as        described in example 1-2, 5-7. The cell sample and the Detection        Molecules are mixed in one container to allow Detection        Molecules to bind to T cells    -   7. Enrichment of MHC molecules with desired characteristics: The        Detection Molecules are enriched for in the same way as        described in example 1-7. Cells associated with Detection        Molecules are sorted by FACS, through means of the        PE-fluorescence signal. The population of cells holding        Detection Molecules are following injected to the FLUIDIGM C1        unit allowing for single cell distribution in a micro-well        system. Other similar devices or platforms for single cell        amplification can also be used.    -   8. Identification of enriched Detection Molecules: For each cell        in the FLUIDIGM C1 unit we will amplify a) the label (=DNA        oligonucleotide barcode) and b) the TCR V-alpha and -beta        chains. In each well specific primers holding cell        identification keys, will be used to amplify DNA oligonucleotide        Label. Likewise specific primers holding cell identification        keys will be used to amplify the TCR-associate genes, the TCR        V-alpha and Beta chains. The complete sequence of the TCR chains        will allow the assembly of a fully functional TCR sequence. In        parallel knowledge about the Binding Molecules associated with        the given T cell, will provide insight o the antigen recognition        of the given TCR. Thus we can obtain paired samples of TCR        sequences and antigen specificities, using the strategy        explained in this example.

Example 10—Results and Conclusions

The expected outcome of this example is knowledge of the TCR receptorcoupled to the knowledge of the recognition of Binding Molecules of thisgiven TCR. Single-cell sorting will enable the generation of a correctlypaired and fully functional TCR. The association of the labels willexplain the recognition motif of this T cell receptor and providevaluable information in this regard. The technique described here willallow the parallel assessment of multiple different T cells whileenabling the capture of specific T cells receptor sequences and havepaired knowledge about the Binding molecules associated to this. Theoverall principle of example 10 is schematized in FIG. 6C.

Example 11

In this example all cells carry the same TCR (T cell receptor). A largenumber of different pMHC complexes (binding molecules) are employed, inorder to examine the recognition breadth and affinity of a given T cellreceptor (TCR).

Example 11 Explained

This example describes how the detection of Binding Molecules can beused to determine the recognition breadth and affinity of a given T cellreceptor (TCR).

This is an example where the Sample (1) is a T cell clone or a cultureof T cell receptor transduced T cells. Importantly for this example allcells will hold the same T cell receptor.

The Linker (2) was a dextran conjugate with streptavidin andfluorochrome (Dextramer backbone from Immudex).

The Binding Molecules (3) will be peptide-MHC (pMHC) complexesdisplaying a library of peptides that are potentially recognized by theT cell receptor in question. This could ideally be a set ofalanine-scanning substituted peptides modified based on the knownrecognition sequence of the T cell receptor in question. The BindingMolecules will be modified (3b) by biotinylation and quality controlledas described in example 1-6.

The Labels (4) are oligonucleotides as in example 7-9.

The detection molecule (5) will be synthetized (5a) and modified (5b) asin example 7-9. Multiple different detection molecules will be generatedwherein the individual detection molecules containing different pMHCwere encoded by corresponding individual oligonucleotide labels, as inexample 7-9.

An amount of Sample, T cell clone (1b) will be mixed with detectionmolecules (5) under conditions (6c) that allow binding of detectionmolecules to the T cells in the sample.

Cells will be washed to remove excess Detection Molecules, and followingthe identity of the Binding Molecules will be revealed throughsequencing of the Label.

This example thus reveals the possibility to identify the specificityand breadth of specificity of a given T cell receptor. This knowledge isessential for the development of T cell receptor gene therapystrategies, and adoptive transfer of T cell population carrying specificT cell receptors.

Example 11 in Detail

-   -   1. Sample preparation. The cell sample to be used in this        example is a T cell clone (carrying a single, defined T cell        receptor) or a culture of T cells transduced with a given T cell        receptor.        -   e. Acquiring sample: Any PBMC sample can be used as a source            to transduce the T cells with a characterized T cell            receptor (TCR), expressed in a retroviral vector.        -   f. Modifying sample: PBMCs modified to express a given TCR            following retroviral transduction using a vector expressing            the selected TCR.    -   2. Linker preparation: The linker used in this example is        prepared as in example 1-10. The fluorochrome has no particular        use in this example. Consequently, the fluorochrome attachment        is not needed.    -   3. Binding molecule preparation: The binding molecules used in        this example will be a collection of peptide-MHC molecules,        designed to assess the breadth to recognition of the given TCR.        The peptide library could be alanine substitution libraries of        the known peptide-epitope recognized by the given TCR. Alanine        substitution is used to assess the essential amino acids in        given positions important for TCR recognition.        -   a. Synthesis: as in example 7-9.        -   b. Modification: No further modifications        -   c. Purification: as in example 7-9.    -   4. Label preparation: as in example 7-9.    -   5. Detection molecules preparation: as in example 7-9.    -   6. Incubation of sample and detection molecules: The cell sample        and the Detection Molecules were mixed in one container, to        allow the Detection Molecules to bind the T cells that they        recognize.        -   a. Amount of sample: 100.000 cells expressing a given TCR        -   b. Amount of detection molecule: as in example 7-9.        -   c. Conditions: The 100.000 cells will be mixed with            different quantities of Detection Molecules. This being the            standard amount of detection molecules as given in example            7-9, but with parallel detection analysis using e.g. 5×            excess for Detection molecules, as well as 5×, 25×, 125×            fold less Detection Molecules. Such titration is done to            assess the avidity of the selected TCR towards the Binding            Molecule library used.    -   7. Enrichment of detection molecules with desired        characteristics: In this example, excess Detection molecules are        separated from Sample through centrifugation. All cells are        expected to bind Detection molecules to some extent, and        consequently all cells are used for characterization of Binding        Molecules.    -   8. Identification of enriched Detection Molecules: The        identification of Detection Molecules and analyses of sequencing        results is conducted as described in example 7.

Example 11—Results and Conclusions

The expected outcome of this example is knowledge related to theDetection molecules associated with a given T cell receptor. Throughthis technology we can gain knowledge on the breadth and the avidity ofa given TCR towards a large library of similar, overlapping and/oralanine substituted peptide-epitope sequences. The technique can be usedto understand both avidity and ‘fine-specificity’ of a given TCR. Thiswill be of crucial importance for development of TCR receptor-associatedtherapies, such as TCR gene therapy in cancer treatment. The overallprinciple of example 11 is schematized in FIG. 6D.

Example 12

In this example it is described how the present invention can be used toidentify the specificity of the stimuli that lead to a functionalresponse of certain cells. In the example, the external stimulus is theaddition of tumor cells, and the functional response measured is therelease of INF-γ.

Example 12 Explained

This example will describe how the detection of Binding Molecules can beassociated to a functional response to a given stimuli provided to theSample in vitro.

This is an example where the Sample (1) will be tumor infiltratinglymphocytes (TIL) from cancer patients which are modified to determinethe reactivity toward tumor cells (1b). The Sample is mixed with tumorcells to allow cytokine release from responding T cells in the Sample.The cytokines are trapped intracellular following Golgi-transportblockade and cellular fixation.

The Linker (2) was a dextran conjugate with streptavidin andfluorochrome (Dextramer backbone from Immudex).

The Binding Molecules (3) will be peptide-MHC (pMHC) complexesdisplaying a library of melanoma-associated peptides (as described inAndersen et al. Dissection of T cell antigen specificity in humanmelanoma. Cancer Research 2012 Apr. 1; 72(7):1642-50), and used inexample 7. The Binding Molecules will be modified (3b) by biotinylationand quality controlled as described in example 1-6.

The Labels (4) were oligonucleotides as in example 7-9.

The detection molecule (5) will be synthetized (5a) and modified (5b) asin example 7-9. 175 different detection molecules were generated whereinthe individual detection molecules containing different pMHC wereencoded by corresponding individual oligonucleotide labels, as inexample 7.

An amount of Sample, TILs (1b) will mixed with detection molecules (5)under conditions (6c) that allow binding of detection molecules to Tcells in the sample. Cells will be selected by FACS based on theircytokine release upon stimulation with the tumor cells. After cellularselection, cells encompassing different cytokine profiles will beanalyzed for their binding to the Detection Molecules, consequentlydescribing the T cell receptor specificity of the responding T cells,i.e. T cells recognizing tumor cells.

This example thus reveal the possibility to identify potentialdifferences in T cell specificity among cells responding differently toa given stimuli, here provided be the mixture of T-cells with tumorcells.

Example 12 in Detail

-   -   1. Sample preparation. The cell sample to be used in this        example is tumor infiltrating lymphocytes (TILs) obtained from        melanoma patients (as example 7).        -   a. Acquiring sample: as in example 7.        -   b. Modifying sample: TILs will be purified and expanded as            in example 7. Following, TILs will be mixed with tumor cell            to assess cytokine release upon T-cell mediated tumor cell            recognition, using the following protocol for intracellular            cytokine staining: Tumor cells will be thawed, washed twice            in culture media (RPMI), and cultured in RPMI+10% FCS (R10)            until they had expanded to a sufficient amount of cells.            TILs will be thawed in 10 ml RPMI+2.5 ul DNase and 50 ul            MgCl2, washed twice in RPMI and rested overnight or at least            4 hr in X-vivo+5% HS+100 U/ml IL-2. Following rest, the            cells will be washed, counted and resuspended in X-vivo+5%            HS obtaining a concentration of 3*10⁶ cells/ml. 100 ul of            the cell suspension will then be added to at least two wells            in a 96 well plate—more replicates will be made if enough            cells are available. Thus 3*10⁵ cells will added to each            well. Tumor cells will be trypsinated, washed in R10,            counted and resuspended in RPMI+10% HS to a concentration of            2*106 cells/ml. 50 ul of cell suspension containing 1*10⁵            cells will be added to the TILs. To every well, 50 ul of            Golgi medium, containing 45 ul RPMI+10% HS, 5 ul BV421            conjugated CD107a antibody (BD pharmingen) and 0.2 ul Golgi            Plug (BD Bioscience 555029) was added. The cells were then            incubated 4-5 hours in 37° C. All samples were cultured in a            ratio of 3:1 TIL:tumor cells, with a total of 4*10⁵ cells            per well. After end incubation, the cells were spun and all            replicates were collected into one well. Cell will be washed            in PBS+2% FCS (FACS buffer) and resuspended in 50 uL            barcode-buffer (PBS/0.5% BSA/2 mM EDTA/100 μg/ml herring            DNA).    -   2. Linker preparation: The linker used in this example is        prepared as in example 1-8. The fluorochrome has no particular        use in this example. Consequently, the fluorochrome attachment        is not needed.    -   3. Binding molecule preparation: The binding molecules used in        this example will be 175 different class I MHC-peptide        complexes, as in example 7.        -   a. Synthesis: as in example 7.        -   b. Modification: No further modifications        -   c. Purification: as in example 7.    -   4. Label preparation: as in example 7.    -   5. Detection molecules preparation: as in example 7.    -   6. Incubation of sample and detection molecules: The cell sample        and the Detection Molecules were mixed in one container, to        allow the Detection Molecules to bind the T cells that they        recognize.        -   a. Amount of sample: 50 uL cell suspension as described in            1b.        -   b. Amount of detection molecule: as in example 7.        -   c. Conditions: The Detection molecules were added in the            required amount. If necessary barcode-buffer was added to            reach a total volume of 100 ul and cells were incubated 15            min, 37° C. Following incubation, cells were and stained            with the following surface antibodies: anti-CD3 antibody,            anti-CD8 antibody, anti-CD4 antibody and near-IR-viability            dye (Invitrogen L10119). The cells were then incubated for            30 min at 4° C., after which they were washed twice in            barcode-buffer and incubated in 200 ul fixation buffer (1:4            concentrate, eBioscience 00-5123-43, to diluent eBioscience            00-5223-56) overnight at 4° C. The following day, cells were            washed twice in permeabilization buffer (1:10 buffer to            water, eBioscience 00-8333-56), resuspended in 50 ul            permeabilization buffer and stained with intracellular            antibodies: FITC-conjugated anti-TNF antibody (BD pharmingen            562082), APC-conjugated anti-IFN antibody (BD 341117). After            incubating for 30 min at 4° C. with the antibodies, the            cells were washed twice in permeabilization buffer,            resuspended in 50 ul barcode-buffer.    -   7. Enrichment of detection molecules with desired        characteristics: In this Example, the Sample is selected based        on the cytokine secretion mediated following incubation with        tumor cells. Cytokines are visualized through intracellular        cytokine staining (ICS). The Sample is following stained with        the Detection Molecules. Cytokine producing cells will be        selected by Fluorescence-Activated-Cell-Sorting (FACS), and the        Detection Molecules carried along with a given cytokine profile        of a given cell population will be assessed through sequencing        of the co-attached oligonucleotide barcodes        -   a. Apply: Cells were sorted on a BD FACSAria, equipped with            three lasers (488 nm blue, 633 nm red and 405 violet). The            flow cytometry data analyses will be performed using the BD            FACSDiva software version 6.1.2. The following gating            strategy will be applied. Lymphocytes were identified in a            FSC/SSC plot. Additional gating on single cells            (FSC-A/FSC-H), live cells (near-IR-viability dye negative),            and CD8, CD3 positive cells, and CD4 negative.            -   From this defined cell population two separate subsets                will be sorted based ion the cytokine secretion in                relation to tumor-cell stimulation. T cells positive for                any of the detected cytokines ‘ICS positive’ is sorted                in one tube, and T cells negative for any of the                detected cytokines ‘ICS negative’ is sorted in another                tube.        -   b. Wash: not applicable.        -   c. Separate: Optionally cells were acquired up to one week            after fixation in 1% paraformaldehyde. The ‘ICS positive’            and ‘ICS negative’ cells were sorted by FACS, as described            in 7a, into tubes that had been pre-saturated for 2 h-O.N.            in 2% BSA and contained 200 μl barcode-buffer to increase            the stability of the oligonucleotides that followed with the            sorted cells. The sorted cells were centrifuged 5 min, 5000            g, to allow removal of all excess buffer. Cells were stored            at −80° C.    -   8. Identification of enriched Detection Molecules: The        identification of Detection Molecules and analyses of sequencing        results is conducted as described in example 7. The ‘ICS        positive’ and ‘ICS negative’ cells are treated as two        independents samples. Following these two samples are compared        for the association with Binding Molecules.

Example 12—Results and Conclusions

The expected outcome of this examples is knowledge related to theDetection molecules associated with tumor cell recognition (=‘ICSpositive’) as oppose to no recognition (=‘ICS negative’). In otherterms, we will gain knowledge on the T cell recognition elementsassociated with tumor cell recognition, among a large pool of differentDetection Molecules, here peptide-MHC molecules. Knowledge of T cellmediated recognition of tumor cells has major impact on the design anddevelopment of immunotherapeutic strategies for cancer. The overallprinciple of example 12 is schematized in FIG. 6A.

Cell could likewise be selected based on e.g. phenotypiccharacteristics, to assess what T specificities are associated withselected phenotypic characteristics as schematized in FIG. 6B.

Tables for Examples 1-12:

TABLE 1 BCs used in Examples 1-7 with indicated frequencies ofpeptide-antigen specific T cells as identified by conventional MHCmultimer staining: Epitope Freq. (%) BC171 A11 EBV-EBNA4 0.32 A3 CMVpp150 TVY 0.015 BC254 A2 FLU MP 58-66 GIL 0.0522 A2 EBV LMP2 FLY 0.014A2 CMV pp65 NLV 1.128 BC261 A2 FLU MP 58-66 GIL 0.125 A3 EBV EBNA 3a RLR0.0258 A2 EBV LMP2 FLY 0.0075 BC266 A1 CMV pp65 YSE 0.0859 A1 FLU BP-VSD0.0628 BC268 A2 FLU MP 58-66 GIL 0.2523 A2 CMV pp65 NLV 0.5445 BC260 A2FLU MP 58-66 GIL 0.0456 A2 CMV pp65 NLV 0.134 B7 CMV pp65 TPR 4.5395BC262 A11 EBV-EBNA4 0.0872

TABLE 2 Structure and sequences of 2OS A oligonucleotides and 2OS Boligonucleotides, used to produce 2OS DNA-barcodes: Oligo name 5′modification Forward primer region 6xN region Coding regionAnnealing region 2OS-1-Oligo-A1 Biotin-C6- GAAGTTCCAGCCAGCGTCACAGTTTNNNNNN CGAGGGCAATGGTTAACTGACACGT GGTCAGCATCATTTCC 2OS-1-Oligo-A2Biotin-C6- GAAGTTCCAGCCAGCGTCACAGTTT NNNNNN CAGAAAGCAGTCTCGTCGGTTCGAAGGTCAGCATCATTTCC 2OS-1-Oligo-A3 Biotin-C6- GAAGTTCCAGCCAGCGTCACAGTTTNNNNNN TAAGTAGCGGGCATAATGTACGCTC GGTCAGCATCATTTCC 2OS-1-Oligo-A4Biotin-C6- GAAGTTCCAGCCAGCGTCACAGTTT NNNNNN GGATCCAGTAAGCTACTGCGTTTATGGTCAGCATCATTTCC 2OS-1-Oligo-A5 Biotin-C6- GAAGTTCCAGCCAGCGTCACAGTTTNNNNNN GGGCTGCGGAGCGTTTACTCTGTAT GGTCAGCATCATTTCC 2OS-1-Oligo-A6Biotin-C6- GAAGTTCCAGCCAGCGTCACAGTTT NNNNNN AAACGTATGTGCTTTGTCGGATGCCGGTCAGCATCATTTCC 2OS-1-Oligo-A7 Biotin-C6- GAAGTTCCAGCCAGCGTCACAGTTTNNNNNN ATATCATCATAGGCTTAGCGACGTA GGTCAGCATCATTTCC 2OS-1-Oligo-A8Biotin-C6- GAAGTTCCAGCCAGCGTCACAGTTT NNNNNN AGGAAAATCTGCTACCGCCAATGATGGTCAGCATCATTTCC 2OS-1-Oligo-A9 Biotin-C6- GAAGTTCCAGCCAGCGTCACAGTTTNNNNNN CTGATTGACTGCATGGAGGCTATAC GGTCAGCATCATTTCC 2OS-1-Oligo-A10Biotin-C6- GAAGTTCCAGCCAGCGTCACAGTTT NNNNNN GTGGCGACTTCACGATTATCTGAACGGTCAGCATCATTTCC 2OS-1-Oligo-A11 Biotin-C6- GAAGTTCCAGCCAGCGTCACAGTTTNNNNNN CCTGTATTGAAGGTTCAGTCCTGTT GGTCAGCATCATTTCC 2OS-1-Oligo-A12Biotin-C6- GAAGTTCCAGCCAGCGTCACAGTTT NNNNNN GGCTCTATAAGGTTTCCTCAAAGGTGGTCAGCATCATTTCC 2OS-1-Oligo-A13 Biotin-C6- GAAGTTCCAGCCAGCGTCACAGTTTNNNNNN TTGGGAGCTTTCCTATGTACAGTCC GGTCAGCATCATTTCC 2OS-1-Oligo-A14Biotin-C6- GAAGTTCCAGCCAGCGTCACAGTTT NNNNNN AGAGAATATGTCGCTCCCGTTATGTGGTCAGCATCATTTCC 2OS-1-Oligo-A15 Biotin-C6- GAAGTTCCAGCCAGCGTCACAGTTTNNNNNN GCAGTTAGATATGCAGTTACCTGAC GGTCAGCATCATTTCC 2OS-1-Oligo-A16Biotin-C6- GAAGTTCCAGCCAGCGTCACAGTTT NNNNNN CTTCACCCGAACATGCAGTGTTATTGGTCAGCATCATTTCC 2OS-1-Oligo-A17 Biotin-C6- GAAGTTCCAGCCAGCGTCACAGTTTNNNNNN AAAGCCGTTGCAGTATCGTCTGAGC GGTCAGCATCATTTCC 2OS-1-Oligo-A18Biotin-C6- GAAGTTCCAGCCAGCGTCACAGTTT NNNNNN GCTGGATGTTAATAACTGCGGTCCGGGTCAGCATCATTTCC 2OS-1-Oligo-A19 Biotin-C6- GAAGTTCCAGCCAGCGTCACAGTTTNNNNNN ACGAGTTGACATGGACGGATCCCTC GGTCAGCATCATTTCC 2OS-1-Oligo-A20Biotin-C6- GAAGTTCCAGCCAGCGTCACAGTTT NNNNNN TTCATCACTCATTGTTCTGAGTAGGGGTCAGCATCATTTCC 2OS-1-Oligo-A21 Biotin-C6- GAAGTTCCAGCCAGCGTCACAGTTTNNNNNN ATGTTTAATCTAACTTGATGCCTCC GGTCAGCATCATTTCC 2OS-1-Oligo-A22Biotin-C6- GAAGTTCCAGCCAGCGTCACAGTTT NNNNNN TAATACGCCTGAGGTGTTGGGTTGCGGTCAGCATCATTTCC 2OS-1-Oligo-B1 CTGTGACTATGTGAGGCTTTCTCGA NNNNNNGCCTGTAGTCCCACGCGATCTAACA GGAAATGATGCTGACC 2OS-1-Oligo-B2CTGTGACTATGTGAGGCTTTCTCGA NNNNNN CAACCATTGATTGGGGACAACTGGGGGAAATGATGCTGACC 2OS-1-Oligo-B3 CTGTGACTATGTGAGGCTTTCTCGA NNNNNNACGTTTAAGCATCTGTACTCCAGAT GGAAATGATGCTGACC 2OS-1-Oligo-B4CTGTGACTATGTGAGGCTTTCTCGA NNNNNN GAATTGAAGCCATCGTTTCGCGCAAGGAAATGATGCTGACC 2OS-1-Oligo-B5 CTGTGACTATGTGAGGCTTTCTCGA NNNNNNCGTAGCTTTTGTAGCGTCTGAGGGC GGAAATGATGCTGACC 2OS-1-Oligo-B6CTGTGACTATGTGAGGCTTTCTCGA NNNNNN AATCGTCAGTCCCTGTTTCGACATCGGAAATGATGCTGACC 2OS-1-Oligo-B7 CTGTGACTATGTGAGGCTTTCTCGA NNNNNNCGGTGGTAGGTGATACTTCTGTACC GGAAATGATGCTGACC 2OS-1-Oligo-B8CTGTGACTATGTGAGGCTTTCTCGA NNNNNN TGACTATCGGGGCGTGACATGAGCTGGAAATGATGCTGACC 2OS-1-Oligo-B9 CTGTGACTATGTGAGGCTTTCTCGA NNNNNNGTTGGTGAAACTACCGACGCTTTAC GGAAATGATGCTGACC 2OS-1-Oligo-B10CTGTGACTATGTGAGGCTTTCTCGA NNNNNN AATGGAGGTGCAGGAATACTCTCGTGGAAATGATGCTGACC 2OS-1-Oligo-B11 CTGTGACTATGTGAGGCTTTCTCGA NNNNNNAAAACGCACCACAACTCGGACGTGA GGAAATGATGCTGACC 2OS-1-Oligo-B12CTGTGACTATGTGAGGCTTTCTCGA NNNNNN GCCATATAGCACAGCACGCAATCCGGAAATGATGCTGACC 2OS-1-Oligo-B13 CTGTGACTATGTGAGGCTTTCTCGA NNNNNNCCTATGCGAACTTGGTTTATCCTGC GGAAATGATGCTGACC 2OS-1-Oligo-B14CTGTGACTATGTGAGGCTTTCTCGA NNNNNN AAGCTGCGTATCCTCGAACTAGCAGGGAAATGATGCTGACC 2OS-1-Oligo-B15 CTGTGACTATGTGAGGCTTTCTCGA NNNNNNATGGCGCAGACATTCTGTAGTCGCA GGAAATGATGCTGACC 2OS-1-Oligo-B16CTGTGACTATGTGAGGCTTTCTCGA NNNNNN CTTATGGACTGGTTGGGGACAATCCGGAAATGATGCTGACC 2OS-1-Oligo-B17 CTGTGACTATGTGAGGCTTTCTCGA NNNNNNGTCCTTCCTTACGAAATATTGGTC GGAAATGATGCTGACC 2OS-1-Oligo-B18CTGTGACTATGTGAGGCTTTCTCGA NNNNNN TGATGAACCAATCCTCCGATTTCTTGGAAATGATGCTGACC 2OS-1-Oligo-B19 CTGTGACTATGTGAGGCTTTCTCGA NNNNNNACCGAATGTGGGCCACGAGTCATTC GGAAATGATGCTGACC 2OS-1-Oligo-B20CTGTGACTATGTGAGGCTTTCTCGA NNNNNN CGGGTGAGCATATAACTTGCAATTCGGAAATGATGCTGACC 2OS-1-Oligo-B21 CTGTGACTATGTGAGGCTTTCTCGA NNNNNNAGAATTGTGCTTGGGGCGATTCATA GGAAATGATGCTGACC 2OS-1-Oligo-B22CTGTGACTATGTGAGGCTTTCTCGA NNNNNN AATTGGTGACATGCTTAACTACCGTGGAAATGATGCTGACC 2OS-1-Oligo-B23 CTGTGACTATGTGAGGCTTTCTCGA NNNNNNGAGACGCCTAGAAAGTGATTAACTC GGAAATGATGCTGACC 2OS-1-Oligo-B24CTGTGACTATGTGAGGCTTTCTCGA NNNNNN ATTACAGTTACAGTGCTGGTCGCAGGGAAATGATGCTGACC 2OS-1-Oligo-B25 CTGTGACTATGTGAGGCTTTCTCGA NNNNNNCGTTACGTTGGTGGGCTCTTGGTAC GGAAATGATGCTGACC 2OS-1-Oligo-B26CTGTGACTATGTGAGGCTTTCTCGA NNNNNN GTTATTATCGGTGTCCCGACTAGTTGGAAATGATGCTGACC 2OS-1-Oligo-B27 CTGTGACTATGTGAGGCTTTCTCGA NNNNNNAGAAATGATTCCCGAGTCGCCTTTT GGAAATGATGCTGACC 2OS-1-Oligo-B28CTGTGACTATGTGAGGCTTTCTCGA NNNNNN TGCTCTCGGATGTGGTTCTATGGATGGAAATGATGCTGACC 2OS-1-Oligo-B29 CTGTGACTATGTGAGGCTTTCTCGA NNNNNNGAGTTAAAACCGTCGCCGTAGCACT GGAAATGATGCTGACC 2OS-1-Oligo-B30CTGTGACTATGTGAGGCTTTCTCGA NNNNNN TGTACGCGATAGTACTCGGGTCCTGGGAAATGATGCTGACC 2OS-1-Oligo-B31 CTGTGACTATGTGAGGCTTTCTCGA NNNNNNAACTTACGCCCAGCAAGGCATTCAT GGAAATGATGCTGACC 2OS-1-Oligo-B32CTGTGACTATGTGAGGCTTTCTCGA NNNNNN AGCATGGCACAAGAGGAGCACTTCAGGAAATGATGCTGACC 2OS-1-Oligo-B33 CTGTGACTATGTGAGGCTTTCTCGA NNNNNNCGATCGTGAGTTTGCAGCGTGACGA GGAAATGATGCTGACC 2OS-1-Oligo-B34CTGTGACTATGTGAGGCTTTCTCGA NNNNNN ACAGCTCCAGCCTCCCTTTGTTTGTGGAAATGATGCTGACC 2OS-1-Oligo-B35 CTGTGACTATGTGAGGCTTTCTCGA NNNNNNAAACCTTTGTTCGGGCGTCTACCAT GGAAATGATGCTGACC 2OS-1-Oligo-B36CTGTGACTATGTGAGGCTTTCTCGA NNNNNN TCTTTCAAAACAGCGGGAGTCATCGGGAAATGATGCTGACC 2OS-1-Oligo-B37 CTGTGACTATGTGAGGCTTTCTCGA NNNNNNGCGGTTTATCCGAATCTCACGCTAA GGAAATGATGCTGACC 2OS-1-Oligo-B38CTGTGACTATGTGAGGCTTTCTCGA NNNNNN GCATATGCTACAGGCTGGGGTGAACGGAAATGATGCTGACC 2OS-1-Oligo-B39 CTGTGACTATGTGAGGCTTTCTCGA NNNNNNGGAGGTCTAAACGTCCGGAGCTATT GGAAATGATGCTGACC 2OS-1-Oligo-B40CTGTGACTATGTGAGGCTTTCTCGA NNNNNN AAGAATAAGATTGCGTGCGCCTTAAGGAAATGATGCTGACC 2OS-1-Oligo-B41 CTGTGACTATGTGAGGCTTTCTCGA NNNNNNCATCATCGTCGTCCAAATATGTGAT GGAAATGATGCTGACC 2OS-1-Oligo-B42CTGTGACTATGTGAGGCTTTCTCGA NNNNNN CACGTGTAGCTGTGGGCCAAGTCTAGGAAATGATGCTGACC 2OS-1-Oligo-B43 CTGTGACTATGTGAGGCTTTCTCGA NNNNNNCAGTTGTCAAATCTCCGCATTGGTA GGAAATGATGCTGACC 2OS-1-Oligo-B44CTGTGACTATGTGAGGCTTTCTCGA NNNNNN ACTGGTAATGCCATTGGTCTAAATGGGAAATGATGCTGACC 2OS-1-Oligo-B45 CTGTGACTATGTGAGGCTTTCTCGA NNNNNNGTCTTTGGTCGTAACGAATCTCCGT GGAAATGATGCTGACC 2OS-1-Oligo-B46CTGTGACTATGTGAGGCTTTCTCGA NNNNNN CTTAGGCATGACGGGGTTGTCCATGGGAAATGATGCTGACC 2OS-1-Oligo-B47 CTGTGACTATGTGAGGCTTTCTCGA NNNNNNCCGGTGAATTTTGGGTGTCCATGTA GGAAATGATGCTGACC 2OS-1-Oligo-B48CTGTGACTATGTGAGGCTTTCTCGA NNNNNN CCTTTATCTCCTCCACCTATAAGGTGGAAATGATGCTGACC 2OS-1-Oligo-B49 CTGTGACTATGTGAGGCTTTCTCGA NNNNNNGATACTATATGACGGCCTGTAATCG GGAAATGATGCTGACC 2OS-1-Oligo-B50CTGTGACTATGTGAGGCTTTCTCGA NNNNNN ATTGGTTGGCCGAAAGACTACATCTGGAAATGATGCTGACC 2OS-1-Oligo-B51 CTGTGACTATGTGAGGCTTTCTCGA NNNNNNCGTAGTTATGGGGTGGGTCACCTGC GGAAATGATGCTGACC 2OS-1-Oligo-B52CTGTGACTATGTGAGGCTTTCTCGA NNNNNN AAGTTTCCAGGCACTGATTCGTTCCGGAAATGATGCTGACC 2OS-1-Oligo-B53 CTGTGACTATGTGAGGCTTTCTCGA NNNNNNTTCCTTATTTCCCGGTTGAGATACA GGAAATGATGCTGACC 2OS-1-Oligo-B54CTGTGACTATGTGAGGCTTTCTCGA NNNNNN AGGTATCATGCGGGCCGAATCTTGGGGAAATGATGCTGACC 2OS-1-Oligo-B55 CTGTGACTATGTGAGGCTTTCTCGA NNNNNNATACCCGTAGGCCAGTACCCTCTCC GGAAATGATGCTGACC

TABLE 3 The reagents used for annealing (left) and elongation (right) ofpartly complementary oligonucleotides. Reagents marked in italic arefrom the Sequenase Version 2.0 DNA Annealing reaction (μl) Elongationreaction (μl) Oligo A (100 μM) 2.6 Annealing reaction 10 μl Oligo B (100μM) 5.4 0.1M DTT 1 μl Sequenase reaction 2 H₂O 0.5 μl buffer Total 10 8xdiluted 2 μl Sequenase polymerase 5x diluted Sequence 2 μl extensionmixture Total 15.5

TABLE 4 Overview of reagents required for production of BindingMolecules produced from 100 μg/ml pMHC exchange reaction. The amounts ofDetection Molecule used for staining 1 × 106-2 × 106 cells in 100 μl arealso specified. Amount Exchanged pMHC/ul per SA conjugate D-biotin End:pMHC staining PE dex 1.32 μl 12.6 μM 44 ug/ml 3 ul

TABLE 5 The components of the antibody mixture added while DetectionMolecules are incubated with sample. The amount listed is for incubationof 1 × 10⁶-2 × 10⁶ cells in 100 ul. Target Conjugate Amount (μl) SourceCD8 PerCP 2 Invitrogen MHCD0831 CD4 FITC 1.25 BD bioscience 345768 CD14FITC 3.13 BD bioscience 345784 CD16 FITC 6.25 BD bioscience 335035 CD19FITC 2.50 BD bioscience 345776 CD40 FITC 1.56 Serotec MCA1590F

TABLE 6 The Master mix applied for recovery of DNA-barcodes by QPCR. Thetemplate was drawn from the residual fluid (5-9.25 ul) containing thesorted cells. Nuclease free H₂O was added to a final volume of 25 ul perPCR Component Volume per sample (μl) Master mix 12.5 Probe/SYBR (10uM/100x) 0.25 (0.1 uM/1x) Forward primer (5 uM) 1.5 (300 nM) Reverseprimer (5 uM) 1.5 (300 nM) Template 5-9.25 Nuclease free H₂O 0-4.25Total 25

TABLE 7 The thermal profile applied for qPCR amplification of barcodesassociated with sorted cells. Temperature (° C.) Time No. of cycles 9510 min 1 95 30 s 60 60 s 40

TABLE 8 Structure and sequences of 1OS oligonucleotides applied asDNA barcodes (1OS-1-Oligo-1 to 1OS-1-Oligo-110) Oligo name 5′ modifForward primer region 6xN Coding region Reverse primer region1OS-1-Oligo-1 Biotin-C6- AGATTCTATAAACTGTGCGGTCCTT NNNNNNTATGAGGACGAATCTCCCGCTTATA GGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-2Biotin-C6- AGATTCTATAAACTGTGCGGTCCTT NNNNNN GGTCTTGACAAACGTGTGCTTGTACGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-3 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GTTTATCGGGCGTGGTGCTCGCATAGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-4 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN CCGATGTTGACGGACTAATCCTGACGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-5 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN TAGTAGTTCAGACGCCGTTAAGCGCGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-6 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN CCGTACCTAGATACACTCAATTTGTGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-7 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GGGGTTCCGTTTTACATTCCAGGAAGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-8 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN TATCCCGTGAAGCTTGAGTGGAATCGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-9 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN CTGACGTGTGAGGCGCTAGAGCATAGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-10 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GGTATGGCACGCCTAATCTGGACACGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-11 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GGATGCATGATCTAGGGCCTCGTCTGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-12 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GAGGTCTTTCATGCGTATAGTCACAGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-13 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GATTCAATATGTGTCGTCTATCCTCGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-14 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GGTAACTGCGCATAGTTGGCTCTATGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-15 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GCGTTTAAGGTCACATCGCATGAATGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-16 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GCCCGGGAAGTGTGAGGATATACCCGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-17 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GCTCTTAAAACTGGTATCACCTGACGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-18 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GGGTGGTTAGTGATTTGCCCGTCACGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-19 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN TAGTTGGTGGGTTTCCCTACCGTGTGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-20 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GGTACAGTAAGTGAGAATCCTCTCTGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-21 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GGTTCTAAGTTTAGCGTAGCCGGTTGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-22 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN CTTTAGGTGGGTGCGATTGCCAGTTGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-23 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GCCACCTTAACACGCGATGATATTGGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-24 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GCTATTACGAGCGCTTGGATCCCGTGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-25 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN TATGTTGTGCCTTACGCCTCGATTAGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-26 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN TTAACCGAACTGACGGCCATCAAGGGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-27 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GGGTACATGCGCCTTACTCCTTGTGGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-28 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN TTCTATTCTAAGCCGGCGGTCATATGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-29 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GCTTGATGCTTTACAAGATCGCGTTGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-30 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN TCCAAGTTAGCTTACTCCATGCCCCGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-31 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN AGAACTATTTCCTGGCTGTTACGCGGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-32 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN TCGGTTTCAAGGATGATCCGCGCTTGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-33 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN AGAGACTGCCCGACACATCTTAGTGGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-34 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN CTGTTAATTAGGCTCGGTCGGCCTAGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-35 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN AGGTAGTCCTATGCGGGCTTTCTCTGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-36 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GGCTTGGACTATAGTCATCGCGTTTGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-37 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN CACTGTTTAACAAGCCCGTCAGTAGGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-38 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN ACGTCGTATTATACCCGCCATGGAAGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-39 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN TGCTTAATTTACGACCGATGCTGCGGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-40 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN TCCATAGATTTCTCCGTGAGTCTTTGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-41 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GTGCCGCAGACATTGCATACGATATGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-42 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GCAGGTCCTAACCCGCAACCATTTAGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-43 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN TGCACCGTTCATATGTTATCGGGACGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-44 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN AGAGACTTACACCCGTAGACGTCGGGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-45 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN ATAAAAGAAACCCTCCGCATTGTGTGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-46 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GGTCCCATCCGAGCAGATTTGACTCGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-47 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN ATGAGCTGTCTCGAACCGAAGGCACGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-48 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN TCGGGCGGTTCAACTTACTGGTAGAGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-49 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GGGGAAATAACGGATGCGCTCTTGAGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-50 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN ACTTCTTCTCGGTCGCATGAGGCTGGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-51 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GGATACATATACGCTCGTCGGGACTGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-52 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN CCGGGAAGTGTCATAACTTGAAGCGGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-53 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN CTCAGCCTGCCTCGCTTCTGATATTGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-54 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN AGGGCCAAGTCGACCTAGATGGCTAGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-55 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GGTAGGGCTACTGTTATCCTCCGTCGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-56 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN CGTACGGCTGGAGAGCTGTATGTGGGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-57 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN ACAGGTTGTATTACTTCGCGCCTTGGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-58 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN CTGGGCTCATTACAAGTGTTGCATAGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-59 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN CTAAGTGGCGCCGATTGTTTGTCCAGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-60 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GTATATTTTGCTCCCGGCGACGAGAGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-61 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GCAATTTGCGCTTGTTCGGCATAGCGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-62 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GAGTCGAATATCCACCACCGTATGGGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-63 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN TTGTGGTTTGGGTCCTCAGAGGAGAGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-64 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GGTACCTAGTCTCGTAATCATAGGAGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-65 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GCGGCATGATCTACCTTAAAGCTTGGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-66 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN CCGGCGCAGAAGTTTGAACGAAAAGGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-67 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN ATGCACTATTTTACGTATCCCGTGCGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-68 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GATAGGGTGACTGCTTTCGCGTACAGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-69 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN TATCTGGTAGACATCTCGGCACAGAGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-70 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN TCGGGGTGCAATAATCACTAGTGCTGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-71 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN TAGTTCTGGCTATACACACTTCGGGGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-72 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GCATAGAGTTACCCGATGGATTCGAGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-73 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GTTCATGGTACAGGCTTCTTTACGGGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-74 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN CGATCTCGGGCCTGGGTTTTGAGTAGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-75 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN ATTATTCGTGACCCAACTCATCAGGGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-76 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN CTGAATGGTGAATAATGCGTTCGCCGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-77 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GCTATTAGTTGCTACCCCAAGAATCGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-78 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN AGAAAGTCTTGGATACACGGCCGGGGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-79 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GTGTGTTCCTATGCACAATTTCATAGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-80 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN TACATGGTAGGGGTCTCCGAACCGTGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-81 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN TAGGGATAACTTTCCTCCCACTTGGGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-82 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN TCTGGTGTCTCACCCATGGGATGTCGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-83 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN TAACGATTTTCTCGCGGGAGTTTCGGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-84 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN CGAGCCTGGTTAGCGCCTACAAGAGGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-85 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN CGTAGTAAGATATGTAGTCCACGTCGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-86 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN TGTTAGTTGCCCCATATCTTTACGCGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-87 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GCTGGATTGTGATTGTCCGGATCCGGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-88 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GGGAGGACTGCGGTTCAGCTTACAAGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-89 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GCGTAGGTCTAGTTCAGATTCTATAGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-90 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GTCTACGTGGTTCTATACCATTCGGGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-91 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN AGGCTTTACTACAATGCGTGGGCTCGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-92 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN AGCTTGCTGTATGGGTCATGTTCCTGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-93 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN TGCTCTAAAGACGCGAGGACTACCTGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-94 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN TGTACATGTCATACTCAAGGCTTTAGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-95 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN TTGACATGTACGCCATTTGGGTCGCGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-96 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GCAATTCAGTACGATCGTGTAGCGGGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-97 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN CGCTGTCCAAAGGTTCTTCGTAACGGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-98 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN TTAGACGAGCAGGTTTCTTGCCTATGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-99 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN TCGTTTGGAGCCGTTCACACATGAAGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-100 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN CTGATCAACTTGCGCCCAGCGTTATGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-101 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GACGATGTTGCCTGTTTTGATACGAGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-102 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GGGTAGTCGTGAGGTGAACTCTTCCGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-103 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN AGCCATTTTACGATTCTATTCGATGGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-104 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GTGGTTTATATAATCCCACCTCCTAGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-105 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GCGAAGAACATCCCGGCATTTCATGGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-106 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GCTGGGACAATGCCGAAAACTCTTCGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-107 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN ATTCCGTACCAACCCGCGTCTTAGAGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-108 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN CTGCAGGAGGCTCTAATGCACTCAAGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-109 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GCGTTCAGCATTCATTACGTCTCACGGTACGGCGCTATCATGTACTCATG 1OS-1-Oligo-110 Biotin-C6-AGATTCTATAAACTGTGCGGTCCTT NNNNNN GCTGAGGAAGCCCAATGTTCAGTACGGTACGGCGCTATCATGTACTCATG

TABLE 9Listing of the 110 combinations of peptide-HLA Binding Molecules andthe respective Labels, 1OS and 2OS DNA-barcodes, that were used to encodethe given specificity of all Detection Molecules applied in experiments 3-6.In the table, HLA code ′A1′ refers to HLA-A0101. HLA code ′A2′refers to HLA-A0201. HLA code ′A3′ refers to HLA-A0301. HLA code ′A11′refers to HLA-A1101. HLA code ′A24′ refers to HLA-A2402. HLA code ′B7′refers to HLA-B0702. HLA 1OS Barcode 2OS Barcode code Peptide Sequence 1A1B1 A1 CMV pp65 YSE YSEHPTFTSQY 2 A1B2 A1 CMV pp50 VTE VTEHDTLLY 3 A1B3A1 FLU BP-VSD VSDGGPNLY 4 A1B4 A11 EBV-EBNA4 AVFDRKSDAK 5 A1B5 A11HCMV pp65 GPISGHVLK 6 A1B6 A11 VP1 DLQGLVLDY 7 A1B7 A11 VP1 VLGRKMTPK 8A1B8 A11 VP1 VTLRKRWVK 9 A1B9 A11 VP1 LVLDYQTEY 10 A1B10 A11 VP1GQEKTVYPK 11 A2B1 A11 VP1 VTFQSNQQDK 12 A2B2 A11 VP1 LKGPQKASQK 13 A2B3A11 VP1 NVASVPKLLVK 14 A2B4 A11 VP1 TSNWYTYTY 15 A2B5 A11 VP1LVLDYQTEYPK 16 A2B6 A11 VP1 TLRKRWVKNPY 17 A2B7 A11 VP1 AVTFQSNQQDK 18A2B8 A11 VP1 PLKGPQKASQK 19 A2B9 A2 VP1 RIYEGSEQL 20 A2B10 A11 VP1SLFSNLMPK 21 A3B1 A2 VP1 KLLVKGGVEV 22 A3B2 A11 VP1 SLINVHYWDMK 23 A3B3A2 HPV E6 29-38 TIHDIILECV 24 A3B4 A2 FLU MP 58-66 GIL GILGFVFTL 25 A3B5A2 EBV LMP2 CLG CLGGLLTMV 26 A3B6 A2 EBV BMF1 GLC GLCTLVAML 27 A3B7 A2EBV LMP2 FLY FLYALALLL 28 A3B8 A2 CMV pp65 NLV NLVPMVATV 29 A3B9 A2EBV BRLF1 YVL YVLDHLIVV 30 A3B10 A2 HPV E7 11-20 YMLDLQPETT 31 A4B1 A2CMVIE1 VLE VLEETSVML 32 A4B2 A2 VP1 GCCPNVASV 33 A4B3 A2 VP1 SITQIELYL34 A4B4 A2 VP1 LQMWEAISV 35 A4B5 A2 VP1 AISVKTEVV 36 A4B6 A2 VP1KMTPKNQGL 37 A4B7 A2 VP1 TVLQFSNTL 38 A4B8 A2 VP1 GLFISCADI 39 A4B9 A2VP1 LLVKGGVEVL 40 A4B10 A2 VP1 ELYLNPRMGV 41 A5B1 A2 VP1 NLPAYSVARV 42A5B2 A2 VP1 TLQMWEAISV 43 A5B3 A2 VP1 QMWEAISVKT 44 A5B4 A2 VP1VVGISSLINV 45 A5B5 A2 VP1 SLINVHYWDM 46 A5B6 A2 VP1 HMFAIGGEPL 47 A5B7A2 VP1 FAIGGEPLDL 48 A5B8 A2 VP1 NLINSLFSNL 49 A5B9 A2 VP1 FLFKTSGKMAL50 A5B10 A2 VP1 ALHGLPRYFNV 51 A6B1 A2 VP1 NLINSLFSNLM 52 A6B2 A2 VP1FLDKFGQEKTV 53 A6B3 A2 VP1 VKGGVEVLSV 54 A6B4 A24 HCMV 248-256AYAQKIFKIL 55 A6B5 A24 EBV LMP2 IYVLVMLVL 56 A6B6 A24 EBV BRLF1TYPVLEEMF 57 A6B7 A24 EBV BMLF1 DYNFVKQLF 58 A6B8 A3 CMV pp150 TTVTTVYPPSSTAK 59 A6B9 A3 FLU NP 265-273 ILR ILRGSVAHK 60 A6B10 A3EBV EBNA 3a RLR RLRAEAQVK 61 A1B11 A3 CMV pp150 TVY TVYPPSSTAK 62 A1B12A3 EBV BRLF1 148-56 RVR RVRAYTYSK 63 A1B13 A3 VP1 ASVPKLLVK 64 A1B14 A3VP1 CCPNVASVPK 65 A1B15 A3 VP1 ITIETVLGR 66 A1B16 A3 VP1 NTLTTVLLD 67A1B17 A3 VP1 ALHGLPRYF 68 A1B18 A3 VP1 VASVPKLLVK 69 A1B19 A3 VP1VSGQPMEGK 70 A1B20 A3 VP1 KASSTCKTPK 71 A2B11 A3 VP1 KTPKRQCIPK 72 A2B12A3 VP1 YTYTYDLQPK 73 A2B13 A3 VP1 PITIETVLGR 74 A2B14 B7 VP1 SVARVSLPM75 A2B15 A3 VP1 NSLFSNLMPK 76 A2B16 A3 VP1 KVSGQPMEGK 77 A2B17 A3 VP1TVYPKPSVAP 78 A2B18 A3 VP1 SLINVHYWDMK 79 A2B19 A3 VP1 GVEVLSVVT 80A2B20 A3 VP1 PLDLQGLVL 81 A3B11 A3 VP1 GLDPQAKAK 82 A3B12 A3 VP1EVWCPDPSK 83 A3B13 A3 VP1 ADIVGFLFK 84 A3B14 A3 VP1 KTSGKMALH 85 A3B15A3 VP1 KMALHGLPR 86 A3B16 A3 VP1 RYFNVTLRK 87 A3B17 A3 VP1 TLRKRWVKN 88A3B18 B7 CMV pp65 TPR TPRVTGGGAM 89 A3B19 B7 CMV pp65 RPH-L RPHERNGFTV90 A3B20 B7 EBV EBNA RPP RPPIFIRLL 91 A4B11 B7 VP1 KPGCCPNVA 92 A4B12 B7VP1 QPIKENLPA 93 A4B13 B7 VP1 LPRYFNVTL 94 A4B14 B7 VP1 MPKVSGQPM 95A4B15 B7 VP1 YPKPSVAPA 96 A4B16 B7 VP1 KPSVAPAAV 97 A4B17 B7 VP1APLKGPQKA 98 A4B18 B7 VP1 APKRKASSTC 99 A4B19 B7 VP1 SVARVSLPML 100A4B20 B7 VP1 YPKTTNGGPI 101 A5B11 B7 VP1 YPKPSVAPAA 102 A5B12 B7 VP1KPGCCPNVASV 103 A5B13 B7 VP1 NPRMGVNSPDL 104 A5B14 B7 VP1 LPAYSVARVSL105 A5B15 B7 VP1 TPTVLQFSNTL 106 A5B16 B7 VP1 LPRYFNVTLRK 107 A5B17 B7VP1 YPVVNLINSLF 108 A5B18 B7 VP1 YPKPSVAPAAV 109 A5B19 B7 VP1KPSVAPAAVTF 110 A5B20 B7 VP1 APKRKASST

TABLE 10 The PCR Master mix applied prior to sequencing of DNA- barcodesassociated with sorted cells. The forward and reverse primer includedadaptors for the sequencing reaction (A-key and P1-key respectively).Moreover the forward primer carried a sample-identification barcode(table 12 and 13). The template was drawn from the residual fluid (10-19ul) containing the sorted cells. Nuclease free H2O was added to a finalvolume of 50 ul per PCR Component Volume per sample (μl) Master mix 25Forward primer (5 μM) 3 (300 nM) Reverse primer (5 μM) 3 (300 nM)Template 10-19 Nuclease free H₂O 0-9 Total 50

TABLE 11 The thermal profile applied for amplification of DNA-barcodesassociated with sorted cells. 36 cycles were applied if >1,000 cellswere sorted while 38 cycles were applied if <1,000 cells were sorted.Temperature (° C.) Time No. of cycles 95 10 min 1 95 30 s 60 45 s 36-3872 30 s 72  4 min 1 4 ∞

TABLE 12Forward and reverse primers applied for amplification of enrichedDetection Molecules prior to sequencing. Primers carry Ion Torrent adaptors, A-key and P1-key. Moreover the forward primer encodes a unique sample-IDbarcode (6xN). Compatible with the 1OS Label system (F1 =forward primer, R1 = reverse primer) Primer name Forward primer region6xN Ion Torrent region (A Key) A-Key 1OS-F1-1 GATTCTATAAACTGTGCGGTCCGAAGAT CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 1OS-F1-2GATTCTATAAACTGTGCGGTCC TCCTGA CCATCTCATCCCTGCGTGTCTCCGACTCAGA-Key 1OS-F1-3 GATTCTATAAACTGTGCGGTCC TGTGGACCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 1OS-F1-4 GATTCTATAAACTGTGCGGTCCCATTTA CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 1OS-F1-5GATTCTATAAACTGTGCGGTCC TTACCC CCATCTCATCCCTGCGTGTCTCCGACTCAGA-Key 1OS-F1-6 GATTCTATAAACTGTGCGGTCC ATTCTCCCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 1OS-F1-7 GATTCTATAAACTGTGCGGTCCAGACCC CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 1OS-F1-8GATTCTATAAACTGTGCGGTCC CGCATG CCATCTCATCCCTGCGTGTCTCCGACTCAGA-Key 1OS-F1-9 GATTCTATAAACTGTGCGGTCC TCCTCGCCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 1OS-F1-10 GATTCTATAAACTGTGCGGTCCATTCCT CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 1OS-F1-11GATTCTATAAACTGTGCGGTCC CGTCGA CCATCTCATCCCTGCGTGTCTCCGACTCAGA-Key 1OS-F1-12 GATTCTATAAACTGTGCGGTCC GCCAATCCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 1OS-F1-13 GATTCTATAAACTGTGCGGTCCATACGG CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 1OS-F1-14GATTCTATAAACTGTGCGGTCC GTCAGA CCATCTCATCCCTGCGTGTCTCCGACTCAGA-Key 1OS-F1-15 GATTCTATAAACTGTGCGGTCC CGAGTTCCATCTCATCCCTGCGTGTCTCCGACTCAG Primer name Ion Torrent (P1 Key)Reverse primer region P1-key 1OS-R1 CCTCTCTATGGGCAGTCGGTGATGAGTACATGATAGCGCCGTAC

TABLE 13Forward and reverse primers applied for amplification of enrichedDetection Molecules prior to sequencing. Primers carry Ion Torrent adaptors, A-key and P1-key. Moreover the forward primer encodes a unique sample-IDbarcode. A-key 2OS-F1-1-15 have a 6xN sample-ID barcode region (applied inexperiment 4 and 6) while A-key 2OS-F1-16-40 have a 8xN sample-ID and a2xN spacer (used in all other experiments applying the 2OS system).Compatible with the 2OS Label system (F1 = forward primer, R1 =reverse primer) 6N 2N Primer name Forward primer region region 8N regionspacer A-Key 2OS-F1-1 GAAGTTCCAGCCAGCGTC CTGGGG A-Key 2OS-F1-2GAAGTTCCAGCCAGCGTC CTCCAC A-Key 2OS-F1-3 GAAGTTCCAGCCAGCGTC CTTACCA-Key 2OS-F1-4 GAAGTTCCAGCCAGCGTC TGGCAG A-Key 2OS-F1-5GAAGTTCCAGCCAGCGTC TGAGTA A-Key 2OS-F1-6 GAAGTTCCAGCCAGCGTC ATTCAGA-Key 2OS-F1-7 GAAGTTCCAGCCAGCGTC TGAGCT A-Key 2OS-F1-8GAAGTTCCAGCCAGCGTC GGCGTG A-Key 2OS-F1-9 GAAGTTCCAGCCAGCGTC AAATTGA-Key 2OS-F1-10 GAAGTTCCAGCCAGCGTC GCTGAC A-Key 2OS-F1-11GAAGTTCCAGCCAGCGTC TTCTTA A-Key 2OS-F1-12 GAAGTTCCAGCCAGCGTC TGGTGGA-Key 2OS-F1-13 GAAGTTCCAGCCAGCGTC GCAGTC A-Key 2OS-F1-14GAAGTTCCAGCCAGCGTC TCGTGA A-Key 2OS-F1-15 GAAGTTCCAGCCAGCGTC TACAGTA-Key 2OS-F1-16 GAAGTTCCAGCCAGCGTC TTGCGTTA TG A-Key 2OS-F1-17GAAGTTCCAGCCAGCGTC CGAGCGAG TG A-Key 2OS-F1-18 GAAGTTCCAGCCAGCGTCCGACTCTG TG A-Key 2OS-F1-19 GAAGTTCCAGCCAGCGTC ATCCGTCC TGA-Key 2OS-F1-20 GAAGTTCCAGCCAGCGTC TTAAACGA TG A-Key 2OS-F1-21GAAGTTCCAGCCAGCGTC TAGCTTTT TG A-Key 2OS-F1-22 GAAGTTCCAGCCAGCGTCCACATGTA TG A-Key 2OS-F1-23 GAAGTTCCAGCCAGCGTC GATAGCCA TGA-Key 2OS-F1-24 GAAGTTCCAGCCAGCGTC ACCTGTTA TG A-Key 2OS-F1-25GAAGTTCCAGCCAGCGTC TGCGAATT TG A-Key 2OS-F1-26 GAAGTTCCAGCCAGCGTCGTACATTT TG A-Key 2OS-F1-27 GAAGTTCCAGCCAGCGTC CTATTGCA TGA-Key 2OS-F1-28 GAAGTTCCAGCCAGCGTC ACGATACA TG A-Key 2OS-F1-29GAAGTTCCAGCCAGCGTC CTTAGCGC TG A-Key 2OS-F1-30 GAAGTTCCAGCCAGCGTCCGGAAACC TG A-Key 2OS-F1-31 GAAGTTCCAGCCAGCGTC GATGTTGG TGA-Key 2OS-F1-32 GAAGTTCCAGCCAGCGTC ATCGGCGT TG A-Key 2OS-F1-33GAAGTTCCAGCCAGCGTC TAGTACGA TG A-Key 2OS-F1-34 GAAGTTCCAGCCAGCGTCGACGTGAT TG A-Key 2OS-F1-35 GAAGTTCCAGCCAGCGTC TGAGCCAA TGA-Key 2OS-F1-36 GAAGTTCCAGCCAGCGTC CCTCGCAG TG A-Key 2OS-F1-37GAAGTTCCAGCCAGCGTC AGATCCAG TG A-Key 2OS-F1-38 GAAGTTCCAGCCAGCGTCTTGGCTGA TG A-Key 2OS-F1-39 GAAGTTCCAGCCAGCGTC GACCGCTA TGA-Key 2OS-F1-40 GAAGTTCCAGCCAGCGTC GAGCTTAA TG A-Key 2OS-F1-41GAAGTTCCAGCCAGCGTC GGACTGGT TG A-Key 2OS-F1-42 GAAGTTCCAGCCAGCGTCTGGGAGTC TG A-Key 2OS-F1-43 GAAGTTCCAGCCAGCGTC GCGATGGC TGA-Key 2OS-F1-44 GAAGTTCCAGCCAGCGTC ACTTGGTT TG A-Key 2OS-F1-45GAAGTTCCAGCCAGCGTC ATACTCAT TG A-Key 2OS-F1-46 GAAGTTCCAGCCAGCGTCTAGTGTCC TG A-Key 2OS-F1-47 GAAGTTCCAGCCAGCGTC GCATATAA TGA-Key 2OS-F1-48 GAAGTTCCAGCCAGCGTC GGCGATTG TG A-Key 2OS-F1-49GAAGTTCCAGCCAGCGTC GGGCTGTA TG A-Key 2OS-F1-50 GAAGTTCCAGCCAGCGTCCGCTATTT TG A-Key 2OS-F1-51 GAAGTTCCAGCCAGCGTC GTACTGCA TGA-Key 2OS-F1-52 GAAGTTCCAGCCAGCGTC TGTCTATG TG A-Key 2OS-F1-53GAAGTTCCAGCCAGCGTC CGAATCAC TG A-Key 2OS-F1-54 GAAGTTCCAGCCAGCGTCCGTCCTAA TG A-Key 2OS-F1-55 GAAGTTCCAGCCAGCGTC ACAAATGG TGA-Key 2OS-F1-56 GAAGTTCCAGCCAGCGTC TCTACTTT TG A-Key 2OS-F1-57GAAGTTCCAGCCAGCGTC AATTCGAG TG A-Key 2OS-F1-58 GAAGTTCCAGCCAGCGTCGAACTCGG TG A-Key 2OS-F1-59 GAAGTTCCAGCCAGCGTC GCGGACGC TGA-Key 2OS-F1-60 GAAGTTCCAGCCAGCGTC CTTGTCCA TG A-Key 2OS-F1-61GAAGTTCCAGCCAGCGTC GTCGCGGT TG A-Key 2OS-F1-62 GAAGTTCCAGCCAGCGTCCAGGTCGT TG A-Key 2OS-F1-63 GAAGTTCCAGCCAGCGTC TCTCATCC TGA-Key 2OS-F1-64 GAAGTTCCAGCCAGCGTC GCTTCGTG TG A-Key 2OS-F1-65GAAGTTCCAGCCAGCGTC CGTGATAA TG A-Key 2OS-F1-66 GAAGTTCCAGCCAGCGTCTTGCTCAC TG A-Key 2OS-F1-67 GAAGTTCCAGCCAGCGTC CGCTCTCC TGA-Key 2OS-F1-68 GAAGTTCCAGCCAGCGTC ATTCTACT TG A-Key 2OS-F1-69GAAGTTCCAGCCAGCGTC AAGGCGTT TG A-Key 2OS-F1-70 GAAGTTCCAGCCAGCGTCGCGGGATT TG Primer name Ion Torrent region (A Key) A-Key 2OS-F1-1CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-2CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-3CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-4CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-5CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-6CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-7CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-8CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-9CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-10CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-11CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-12CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-13CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-14CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-15CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-16CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-17CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-18CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-19CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-20CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-21CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-22CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-23CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-24CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-25CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-26CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-27CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-28CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-29CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-30CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-31CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-32CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-33CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-34CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-35CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-36CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-37CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-38CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-39CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-40CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-41CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-42CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-43CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-44CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-45CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-46CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-47CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-48CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-49CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-50CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-51CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-52CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-53CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-54CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-55CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-56CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-57CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-58CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-59CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-60CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-61CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-62CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-63CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-64CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-65CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-66CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-67CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-68CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-69CCATCTCATCCCTGCGTGTCTCCGACTCAG A-Key 2OS-F1-70CCATCTCATCCCTGCGTGTCTCCGACTCAG

TABLE 14Listing of the 175 combinations of peptide-HLA Binding Moleculesand the respective 2OS DNA-barcodes that were used to encode the givenspecificity of all Detection Molecules applied in experiments 7.168 of the peptide-ligands are associated with melanoma while the remaining 7are different virus specific peptides. All are HLA-A0201 peptide antigens.Barcode HLA 2OS allele Peptide Sequence A7B1 HLA-A0201 707-AP RVAALARDAPA7B2 HLA-A0201 ATIC (AICRT) RLDFNLIRV A7B3 HLA-A0201 ATIC (AICRT)MVYDLYKTL A7B4 HLA-A0201 BA46 (MFGE8) NLFETPVEA A7B5 HLA-A0201BA46 (MFGE8) GLQHWVPEL A7B6 HLA-A0201 Bcl-2 PLFDFSWLSL A7B7 HLA-A0201Bcl-2 WLSLKTLLSL A7B8 HLA-A0201 Bcl-xL YLNDHLEPWI A7B9 HLA-A0201 BING-4CQWGRLWQL A7B10 HLA-A0201 B-RAF LATEKSRWSG A7B11 HLA-A0201cyclophilin B (Cyp-B) VLEGMEVV A7B12 HLA-A0201 Cadherin 3/P-cadherinFILPVLGAV A7B13 HLA-A0201 Cadherin 3/P-cadherin FIIENLKAA A7B14HLA-A0201 CDCA1/NUF2 YMMPVNSEV A7B15 HLA-A0201 CDCA1/NUF2 KLATAQFKIA7B16 HLA-A0201 CDK4 ACDPHSGHFV A7B17 HLA-A0201 CML28 (EXOSC5) ALVDAGVPMA7B18 HLA-A0201 COA-1 (UBXN11) FMTRKLWDL A7B19 HLA-A0201 COA-1 (UBXN11)RLLASLQDL A7B20 HLA-A0201 CPSF KVHPVIWSL A7B21 HLA-A0201 CPSF LMLQNALTTMA7B22 HLA-A0201 Cyclin 1 LLDRFLATV A7B23 HLA-A0201 cyclin B1 AGYLMELCCA7B24 HLA-A0201 cyclin B1 AKYLMELTM A8B1 HLA-A0201 B-RAF LATEKSRWS A8B2HLA-A0201 cyclophilin B (Cyp-B) KLKHYGPGWV A8B3 HLA-A0201DAM-6, -10 (MAGE-B1, FLWGPRAYA -B2) A8B4 HLA-A0201 EphA2 IMNDMPIYM A8B5HLA-A0201 EphA2 VLAGVGFFI A8B6 HLA-A0201 EphA2 VLLLVLAGV A8B7 HLA-A0201EphA2 TLADFDPRV A8B8 HLA-A0201 EZH2 FINDEIFVEL A8B9 HLA-A0201 EZH2FMVEDETVL A8B10 HLA-A0201 GnTV VLPDVFIRCV A8B11 HLA-A0201 gp100/Pme117YLEPGPVTA A8B12 HLA-A0201 gp100/Pme117 LLDGTATLRL A8B13 HLA-A0201gp100/Pme117 ITDQVPFSV A8B14 HLA-A0201 gp100/Pme117 VLYRYGSFSV A8B15HLA-A0201 gp100/Pme117 RLMKQDFSV A8B16 HLA-A0201 gp100/Pme117 RLPRIFCSCA8B17 HLA-A0201 gp100/Pme117 AMLGTHTMEV A8B18 HLA-A0201 gp100/Pme117SLADTNSLAV A8B19 HLA-A0201 gp100/Pme117 KTWGQYWQV A8B20 HLA-A0201HERV-K-MEL MLAVISCAV A8B21 HLA-A0201 hsp70 LLLLDVAPL A8B22 HLA-A0201IDO1 ALLEIASCL A8B23 HLA-A0201 LAGE-1 MLMAQEALAFL A8B24 HLA-A0201Livin (ML-IAP) RLASFYDWLP A9B1 HLA-A0201 Livin (ML-IAP) SLGSPVLGL A9B2HLA-A0201 Livin (ML-IAP) QLCPICRAPV A9B3 HLA-A0201 M2BP RIDITLSSV A9B4HLA-A0201 MAGE-A1 KVLEYVIKV A9B5 HLA-A0201 GnTV VLPDVFIRC A9B6 HLA-A0201gp100/Pme117 IMDQVPFSV A9B7 HLA-A0201 gp100/Pme117 MLGTHTMEV A9B8HLA-A0201 hsp70 LLDVAPLSL A9B9 HLA-A0201 MAGE-A10 GLYDGMEHL A9B10HLA-A0201 MAGE-A2 LVHFLLLKY A9B11 HLA-A0201 MAGE-A2 LVQENYLEY A9B12HLA-A0201 MAGE-A2 YLQLVFGIEV A9B13 HLA-A0201 MAGE-A2 KMVELVHFL A9B14HLA-A0201 MAGE-A3 LVFGIELMEV A9B15 HLA-A0201 MAGE-A4 GVYDGREHTV A9B16HLA-A0201 MAGE-A1 YLEYRQVPV A9B17 HLA-A0201 MAGE-A8 GLMDVQIPT A9B18HLA-A0201 MAGE-A8 KVAELVRFL A9B19 HLA-A0201 MAGE-A9 ALSVMGVYV A9B20HLA-A0201 MAGE-C2 ALKDVEERV A9B21 HLA-A0201 MAGE-C2 LLFGLALIEV A9B22HLA-A0201 MAGE-C2 VIWEVLNAV A9B23 HLA-A0201 MAGE-C2 TLDEKVAELV A9B24HLA-A0201 MAGE-C2 KVLEFLAKL A10B1 HLA-A0201 MAGE-A3 KVAELVHFL A10B2HLA-A0201 MC1R TILLGIFFL A10B3 HLA-A0201 Melan-A/MART-1 ELAGIGILTV A10B4HLA-A0201 Melan-A/MART-1 ILTVILGVL A10B5 HLA-A0201 Meloe-1 TLNDECWPAA10B6 HLA-A0201 MG50 CMHLLLEAV A10B7 HLA-A0201 MG50 VLSVNVPDV A10B8HLA-A0201 NY-ESO-1/LAGE-2 QLSLLMWIT A10B9 HLA-A0201 NY-ESO-1/LAGE-2SLLMWITQCFL A10B10 HLA-A0201 P Polypeptide IMLCLIAAV A10B11 HLA-A0201p53 VVPCEPPEV A10B12 HLA-A0201 p53 KTCPVQLWV A10B13 HLA-A0201 p53RMPEAAPPV A10B14 HLA-A0201 p53 LLPENNVLSPV A10B15 HLA-A0201 p53KLCPVQLWV A10B16 HLA-A0201 p53 SMPPPGTRV A10B17 HLA-A0201 p53 LLGRNSFEVA10B18 HLA-A0201 p53 GLAPPQHLIRV A10B19 HLA-A0201 p53 SLPPPGTRV A10B20HLA-A0201 p53 YLGSYGFRL A10B21 HLA-A0201 PGK1 IIGGGMAFT A10B22 HLA-A0201PRAME ALYVDSLFFL A10B23 HLA-A0201 PRAME SLLQHLIGL A10B24 HLA-A0201 SOX10SAWISKPPGV A11B1 HLA-A0201 PRAME SLYSFPEPEA A11B2 HLA-A0201 PRAMEVLDGLDVLL A11B3 HLA-A0201 PRDX5 LLLDDLLVSI A11B4 HLA-A0201NY-ESO-1/LAGE-2 SLLMWITQC A11B5 HLA-A0201 RAB38/NY-MEL-1 VLHWDPETV A11B6HLA-A0201 RAGE-1 LKLSGVVRL A11B7 HLA-A0201 RAGE-1 PLPPARNGGL A11B8HLA-A0201 Replication protein A YLMDTSGKV A11B9 HLA-A0201 SART-3LLQAEAPRL A11B10 HLA-A0201 SART-3 RLAEYQAYI A11B11 HLA-A0201 secernin 1KMDAEHPEL A11B12 HLA-A0201 SOX10 AWISKPPGV A11B13 HLA-A0201 SSX-2RLQGISPKI A11B14 HLA-A0201 SSX-2 KASEKIFYV A11B15 HLA-A0201STAT1-alpha/11 KLQELNYNL A11B16 HLA-A0201 STEAP1 FLYTLLREV A11B17HLA-A0201 STEAP1 LLLGTIHAL A11B18 HLA-A0201 STEAP1 MIAVFLPIV A11B19HLA-A0201 Survivin LMLGEFLKL A11B20 HLA-A0201 Survivin ELTLGEFLKL A11B21HLA-A0201 Survivin TLPPAWQPFL A11B22 HLA-A0201 TAG-1 SLGWLFLLL A11B23HLA-A0201 Telomerase RLFFYRKSV A11B24 HLA-A0201 TRP-2 VYDFFW/LHY A12B1HLA-A0201 NY-ESO-1/LAGE-2 SLLMWITQA A12B2 HLA-A0201 Telomerase RLVDDFLLVA12B3 HLA-A0201 Telomerase ILAKFLHWL A12B4 HLA-A0201 Topoisomerase IIFLYDDNQRV A12B5 HLA-A0201 TRAG-3 ILLRDAGLV A12B6 HLA-A0201 TRP-2FW/LHYYSV A12B7 HLA-A0201 TRP-2 SLDDYNHLV A12B8 HLA-A0201 TRP-2TLDSQVMSL A12B9 HLA-A0201 TRP-2 SVYDFFVWL A12B10 HLA-A0201 TRP2-6bATTNILEHY A12B11 HLA-A0201 tyrosinase CLLWSFQTSA A12B12 HLA-A0201tyrosinase MLLAVLYCL A12B13 HLA-A0201 tyrosinase YMDGTMSQV A12B14HLA-A0201 XBP-1 LLSGQPASA A12B15 HLA-A0201 MG50 LLLEAVPAV A12B16HLA-A0201 MG50 TLKCDCEIL A12B17 HLA-A0201 MG50 WLPKILGEV A12B18HLA-A0201 MG50 RLGPTLMCL A12B19 HLA-A0201 Meloe-2 RCPPKPPLA A12B20HLA-A0201 PRDX5 AMAPIKVRL A12B21 HLA-A0201 cyclin B1 ILIDWLVQV A12B22HLA-A0201 Melan-A/MART-1 EAAGIGILTV A12B23 HLA-A0201 adipophilinSVASTITGV A12B24 HLA-A0201 alpha-actinin-4 FIASNGVKLV A13B1 HLA-A0201Meloe-2 RLPPKPPLA A13B2 HLA-A0201 CDKN1A LMAGCIQEA A13B3 HLA-A0201CDKN1A GLGLPKLYL A13B4 HLA-A0201 CDKN1A FAWERVRGL A13B5 HLA-A0201CLP (coactosin-like protein) NLVRDDGSAV A13B6 HLA-A0201CLP (coactosin-like protein) RLFAFVRFT A13B7 HLA-A0201CLP (coactosin-like protein) VVQNFAKEFV A13B8 HLA-A0201 c-MET YVDPVITSIA13B9 HLA-A0201 CYP1B1 WLQYFPNPV A13B10 HLA-A0201 IMP-3 NLSSAEVVV A13B11HLA-A0201 IMP-3 RLLVPTQFV A13B12 HLA-A0201 KIF20A LLSDDDVVV A13B13HLA-A0201 KIF20A CIAEQYHTV A13B14 HLA-A0201 KIF20A AQPDTAPLPV A13B15HLA-A0201 MAGE-A10 SLLKFLAKV A13B16 HLA-A0201 MAGE-A12 FLWGPRALV A13B17HLA-A0201 MAGE-C2 FLAKLNNTV A13B18 HLA-A0201 Melan-A/MART-1 AAGIGILTVA13B19 HLA-A0201 Survivin QMFFCFKEL A13B20 HLA-A0201 TelomeraseLLTSRLRFI A13B21 HLA-A0201 TYMS LMALPPCHAL A13B22 HLA-A0201FLU MP 58-66 GIL GILGFVFTL A13B23 HLA-A0201 EBV LMP2 CLG CLGGLLTMVA13B24 HLA-A0201 EBV BMF GLC GLCTLVAML A14B1 HLA-A0201 cyclin D1LLGATCMFV A14B2 HLA-A0201 HIV pol TPRVTGGGAM A14B3 HLA-A0201EBV LMP2 FLY FLYALALLL A14B4 HLA-A0201 CMV pp65 NLV NLVPMVATV A14B5HLA-A0201 EBV BRLF1 YVL YVLDHLIVV A14B6 HLA-A0201 BAP31 KLDVGNAEV A14B7HLA-A0201 CMV 1E1 VLE VLEETSVML

Example 20

This is an example where the Sample is a whole blood sample. Instead ofbeing a mix of two PBMC donor materials as described in e.g. example 3it is a mix of two whole blood samples. Except for the samplepreparation the example is performed as example 3

Thus, the Linker is a dextrane conjugate with streptavidin andfluorocrome (PE Dextramer backbone from Immudex ApS).

The Binding Molecules are peptide-MHC (pMHC) complexes. In this examplea panel of 110 labeled pMHC-multimers, constituting a library ofDetection Molecules, are tested. Apart from 26 virus epitopes that arecommonly found in healthy donors (derived from EBV, CMV, FLU and HPV),is included a number of polyomavirus capsid protein (VP1)-derivedepitopes that has previously led to detection of T cells in healthydonors.

110 different Labels are generated as example 3. Detection Molecules aresynthetized as in example 3.

Sample, whole blood, was incubated with an amount of a library ofdetection molecules as described in example 3.

The cell-bound detection molecules are separated from the non-cell bounddetection molecules as described in example 3.

FACS isolated cells were subjected to PCR amplification of theoligonucleotide label associated with the detection molecules bound tocells. Subsequent extensive sequencing of PCR products revealed theidentity of Detection Molecules that bound to the T cells present in thesample.

-   -   1. Sample preparation. The cell samples used in this experiment        are obtained by mixing whole blood from two different donors to        obtain a cell sample where a number of T-cell specificities are        known prior to the experiment. Thus, the sensitivity of the        method as well as the relevance of the results obtained in the        experiment can be evaluated at the end of the experiment, by        comparison with data obtained previously, using other methods        but cell samples prepared from the same donors.        -   a. Acquiring sample: Blood is obtained from the Danish Blood            Bank.        -   b. Modifying sample:            -   i. Blood is drawn into BD Vacutainer® Plus Plastic                K2EDTA Tubes according to manufacturer's protocol.            -   ii. Anti-coagulated blood samples are diluted 1:1 in                RPMI (RPMI 1640, GlutaMAX, 25 mM Hepes; gibco-Life                technologies).            -   iii. The samples used in the experiment are obtained by                mixing blood from a donor (e.g. BC260) with 5% of the T                cells specific for HLA-B7/CMV pp65 TPR into blood from a                donor (e.g. BC262) without HLA-B7/CMV pp65 TPR specific                T cells in fivefold dilutions, creating seven samples                (with 5%, 1%, 0.2%, 0.04%, 0.008%, 0.0016% and 0.00032%                HLA-B7/CMV pp65 TPR-specific T cells). The CMV pp65 TPR                negative sample preparation instead has a population of                HLA-A11/EBV-EBNA4 specific T cells.            -   iv. The remaining of the example is as for example 3.    -   2. Linker preparation: The linker used in this example is        dextrane conjugate with streptavidin and fluorocrome (PE        Dextramer backbone from Immudex) as described in 1.    -   3. Binding molecules preparation: The 110 different Binding        Molecules are prepared as in example 3.    -   4. Label preparation: The 110 different DNA oligo Labels are        prepared as in example 3.    -   5. Detection molecules preparation: 110 different Detection        Molecules are prepared as in example 3.    -   6. Incubation of sample and detection molecules:        -   a. Amount of sample: 0.2 mL anti-coagulated blood diluted            1:1 in RPMI.        -   b. Amount of detection molecule: As for e.g. example 3.        -   c. Conditions: Sample is incubated with dasatinib (50 nM            final concentration), 30 min, 37° C. Subsequently, sample            and Detection Molecule is mixed and incubated as e.g.            example 3.    -   7. Enrichment of detection molecules with desired        characteristics: Detection Molecules are isolated with their        associated cells by FACS as described in example 3.    -   8. Identification of enriched detection molecule: By identifying        the Label (in this Example, the oligonucleotide label), the        pMHCs that bound separated cells can be identified. Therefore,        the oligonucleotide labels that were comprised within the        Detection Molecule that were recovered with the cells, were        sequenced. This allowed the identification of pMHCs that bound        cells of the cell sample. Labels associated with FACS isolated        cells are sequenced as described in example 3.

Example 21

This is an example where the Sample is mononuclear cells derived frombone marrow (BM MNCs). Instead of being a mix of two PBMC donormaterials as described in example 3 it is a mix of two whole bloodsamples. Except for the sample preparation the example is performed asexample 3.

Thus, the Linker is a dextrane conjugate with streptavidin andfluorocrome (PE Dextramer backbone from Immudex).

The Binding Molecules are peptide-MHC (pMHC) complexes. In this examplea panel of 110 labeled pMHC-multimers, constituting a library ofDetection Molecules, are tested. Apart from 26 virus epitopes that arecommonly found in healthy donors (derived from EBV, CMV, FLU and HPV),is included a number of polyomavirus capsid protein (VP1)-derivedepitopes that has previously led to detection of T cells in healthydonors.

110 different Labels are generated as in example 3. Detection Moleculesare synthetized as in example 3.

Sample, whole blood, was incubated with an amount of a library ofdetection molecules as described in example 3.

The cell-bound detection molecules are separated from the non-cell bounddetection molecules as described in example 3.

FACS isolated cells were subjected to PCR amplification of theoligonucleotide label associated with the detection molecules bound tocells. Subsequent extensive sequencing of PCR products revealed theidentity of Detection Molecules that bound to the T cells present in thesample.

-   -   1. Sample preparation. The cell samples used in this experiment        was obtained by mixing bone marrow from two different donors to        obtain a cell sample where a number of T-cell specificities were        known prior to the experiment. Thus, the sensitivity of the        method as well as the relevance of the results obtained in the        experiment could be evaluated at the end of the experiment, by        comparison with data obtained previously, using other methods        but cell samples prepared from the same donors.        -   a. Acquiring sample: Collect bone marrow from the upper            iliac crest or the sternum by using an aspiration needle.        -   b. Modifying sample:            -   i. Dilute aspirated human bone marrow at a ratio of 7:1                with buffer (phosphate buffered saline (PBS), pH 7.2,                and 2 mM EDTA), e.g., dilute 30 mL of bone marrow with 5                mL of buffer to a final volume of 35 mL.            -   ii. Pass cells through a 100 μm filter to remove bone                fragments and cell clumps.            -   iii. Carefully layer 35 mL of diluted cell suspension                over 15 mL of Ficoll-Paque in a 50 mL conical tube.                Centrifuge at 445×g for 35 minutes at 20° C. in a                swinging bucket rotor without brake. Aspirate the upper                layer leaving the mononuclear cell layer undisturbed at                the interphase. Carefully transfer the BM MNCs at the                interphase to a new 50 mL conical tube. Wash cells by                adding up to 40 mL of buffer, mix gently and centrifuge                at 300×g for 10 minutes at 20° C. Carefully remove                supernatant completely. For removal of platelets,                resuspend the cell pellet in 50 mL of buffer and                centrifuge at 200×g for 10-15 minutes at 20° C.                Carefully remove the supernatant completely. Resuspend                cell pellet in 5 mL buffer for downstream applications.            -   iv. The samples used in the experiment is obtained by                mixing BM MNCs from a donor (e.g. BC260) with 5% of the                T cells specific for HLA-B7/CMV pp65 TPR into BM MNCs                from a donor (e.g. BC262) without HLA-B7/CMV pp65 TPR                specific T cells in fivefold dilutions, creating seven                samples (with 5%, 1%, 0.2%, 0.04%, 0.008%, 0.0016% and                0.00032% HLA-B7/CMV pp65 TPR-specific T cells). The CMV                pp65 TPR negative BM MNC preparation instead has a                population of HLA-A11/EBV-EBNA4 specific T cells.            -   v. The remaining of the example is as for example 3.    -   2. Linker preparation: The linker used in this example is        dextrane conjugate with streptavidin and fluorocrome (PE        Dextramer backbone from Immudex) as described in 1.    -   3. Binding molecules preparation: The 110 different Binding        Molecules are prepared as in example 3.    -   4. Label preparation: The 110 different DNA oligo Labels are        prepared as in example 3.    -   5. Detection molecules preparation: 110 different Detection        Molecules are prepared as in example 3.    -   6. Incubation of sample and detection molecules:        -   a. Amount of sample: 0.2 mL anti-coagulated blood diluted            1:1 in RPMI.        -   b. Amount of detection molecule: As for example 3.        -   c. Conditions: Sample is incubated with dasatinib (50 nM            final concentration), 30 min, 37° C. Subsequently, sample            and Detection Molecule is mixed and incubated as e.g.            example 3.    -   7. Enrichment of detection molecules with desired        characteristics: Detection Molecules are isolated with their        associated cells by FACS as described in example 3.    -   8. Identification of enriched detection molecule: By identifying        the Label (in this Example, the oligonucleotide label), the        pMHCs that bound separated cells can be identified. Therefore,        the oligonucleotide labels that were comprised within the        Detection Molecule that were recovered with the cells, were        sequenced. This allowed the identification of pMHCs that bound        cells of the cell sample. Labels associated with FACS isolated        cells are sequenced as described in example 3.

Example 60

This is an example where the binding molecules (BM) are MHC-likeantigen-presenting molecules such as e.g. CD1a, CD1b, CD1c and CD1d.

The Linker used in this example is dextran conjugate with streptavidinand fluorocrome (PE Dextramer backbone from Immudex), the label used isa DNA oligonucleotide and the Sample is PBMC's.

Isolation and identification of detection molecules capable of bindingto cells of the cell sample is done by FACS of cells with PE labeleddextran conjugate, and the identity and amount of associated labels aredetermined by DNA sequencing.

-   -   1. Sample preparation.    -   The sample used in this example can be any type of cell sample,        for example one PBMC sample from a human being, as described in        Example 1.    -   2. Linker preparation:    -   The linker used in this experiment is a dextran molecule,        prepared as described in Example 1.    -   3. Binding molecules preparation        -   a. Synthesis: CD1a, CD1b, CD1c and CD1d is produced as            described by Khurana A, et. al, J Vis Exp. 2007; (10): 556.            and as performed by a person skilled in the art.        -   b. Modification: CD1a, CD1b, CD1c and CD1d is loaded with 15            potential lipid antigens (GMM, glucose monomycolate;            Sulfolipid, diacylated sulfoglycolipid; PIM's,            phosphatidylinositol mannosides; Man-LAM, mannosylated            lipoarabinomannan; MPM, mannosyl-b1-phosphomycoketide; MPP,            mannosyl-b1-phosphoheptaprenol; DDM, dideoxymycobactin;            GSL-1, a-glucoronsylceramide; GaIDAG,            a-galactosyldiacylglycerol; LPG, lipophosphoglycan; PI,            phosphatidylinositol; PG, phosphatidylglycerol; PE,            phosphatidylethanolamine; iGb3, isoglobotrihexosylceramide;            Alpha-GC, a-galactosylceramide). Lipids are dissolved to 2            mg/mL in DMSO, heated to 50° C. for 2 min and diluted            further 1:10 in PBS+0.1% Tween20. Lipids are mixed            individually with the four different CD1 molecules giving            rise to 4×15=60 combinations. Briefly 10 uL 1 ug/uL CD1            protein is mixed with 2 uL 0.2 mg mL lipid in PBS+0.1%            Tween20+10% DMSO. Load lipids into CD1 4 h at 30° C.        -   c. Purification: The CD1a-lipid, CD1b-lipid, CD1c-lipid and            CD1d-lipid complexes are not purified further.    -   4. Label preparation: Labels as described in example 3 are used.        -   a. Synthesis: The first 60 Labels in table 8 are used.        -   b. Modification: no further        -   c. Purification: no further    -   5. Detection molecules preparation        -   a. Synthesis: The detection molecules are prepared as in            example 3 except that the 60 different CD1 and lipid            combinations are mixed with 60 individual DNA            oligonucleotide labels (labels 1-60 from table 8).        -   b. Modification: no further modifications        -   c. Purification: no further    -   6. Incubation of sample and detection molecules        -   The sample and detection molecules of step 1 and 5,            respectively, are mixed and incubated as described in            Example 3.    -   7. Enrichment of detection molecules with desired        characteristic: Cells positive for PE fluorochrome on the Linker        is isolated by FACS as described in example 3.    -   8. Identification of enriched detection molecule: The detection        molecules recovered in step 7 are identified by sequencing, as        described in Example 3.

Example 61

This is an example where the binding molecules (BM) are MHC class IIproteins. The Linker used in this example is dextran conjugate withstreptavidin and fluorocrome (PE Dextramer backbone from Immudex), thelabel used is a DNA oligonucleotide and the Sample is PBMC's.

Isolation and identification of detection molecules capable of bindingto cells of the cell sample is done by FACS of cells with PE labeleddextran conjugate, and the identity and amount of associated labels aredetermined by DNA sequencing.

-   -   1. Sample preparation: The sample used in this example can be        any type of cell sample, for example one PBMC sample from a        human being, as described in Example 1.    -   2. Linker preparation: The linker used in this experiment is a        dextran molecule, prepared as described in Example 1.    -   3. Binding molecules preparation        -   a. Synthesis: MHC class II protein in the form of            biotinylated monomers are obtained from The NIH Tetramer            Core Facility, at Emory University, US. The following eight            MHC Class II monomers are used (DPB1*04:01 C. tetani TT            948-968 FNNFTVSFWLRVPKVSASHLE, DPB1*04:01 human MAGE3            243-258 KKLLTQHFVQENYLEY, DPB1*04:01 human oxytocinase            272-284 KKYFAATQFEPLA, DPB1*04:01 human CLIP 87-101            PVSKMRMATPLLMQA, DPB1*04:01 human CTAG1 157-170            SLLMWITQCFLPVF, DPB1*04:01 HIV env 31-45 TEKLVVVTVYYGVPVW,            DQB1*03:02 human CLIP 87-101 PVSKMRMATPLLMQA, DQB1*03:02            human FcR2 104-119 QDLELSWNLNGLQADL        -   b. Modification: Monomers are obtained ready folded and            biotinylated from The NIH Tetramer Core Facility.        -   c. Purification: No further modification    -   4. Label preparation        -   a. Synthesis: The first 8 labels from Table 8 are used        -   b. Modification: No further        -   c. Purification: No further    -   5. Detection molecules preparation        -   a. Synthesis: The detection molecules are prepared as in            example 3 except that the 8 different MHC class II molecules            are combined with 8 different DNA oligonucleotide labels as            described in example 3.        -   b. Modification: No further        -   c. Purification: No further    -   6. Incubation of sample and detection molecules: The sample and        detection molecules of step 1 and 5, respectively, are mixed and        incubated as described in Example 3.    -   7. Enrichment of detection molecules with desired        characteristics: Cells positive for PE fluorochrome on the        Linker is isolated by FACS as described in example 5.    -   8. Identification of enriched detection molecule: The detection        molecules recovered in step 7 are identified by sequencing, as        described in Example 3.

Example 79

In this example, 20 different pMHC complexes (e.g. corresponding to 20known cancer epitopes) are used as binding molecules, the linker isdextran-streptavidin-PE conjugate, and 20 different DNA oligonucleotidesare used as labels.

The detection of detection molecules capable of binding to cells of thesample, is by immobilization on an array of anti-sense DNAoligonucleotides.

20 different pMHC complexes (all A*02-01, but each of the 20 complexescarrying one specific peptide epitope commonly found in cancer patients)(e.g epitopes from antigens such as WT-1, survivin, and NY-ESO-1) areprepared using standard procedures, and as described in other examplesabove. The pMHC complexes are preferably mono-biotinylated.

Dextran-streptavidin-PE conjugate is used as linker, as described inseveral examples above. PE is a strong fluorochrome, and a givenconjugate carries e.g. 1-3 PE molecules.

The labels are 20 different DNA oligonucleotides of different sequences,of approximately 50 nt in length. The oligonucleotides are biotinylatedat one terminus. Detection molecules are generated by mixing thedextran-streptavidin-PE conjugate with the 20 different biotinylatedpMHC complexes and the 20 different biotinylated DNA oligonucleotidesfrom above, using standard conditions, as described in several examplesabove. This leads to generation of 20 detection molecules, each of whichcarries a specific pMHC complex and a specific DNA label.

Whole blood from e.g. a cancer patient is then mixed with the detectionmolecules generated immediately above, and incubated for 15 minutes.Then the cells are spun down and supernatant removed, and thecentrifugation and resuspension repeated 0-3 times. Finally, the cellsare resuspended in an appropriate binding buffer that allows specifichybridization of complementary DNA strands.

After incubation and washing the solution of cells and detectionmolecules is applied to an array, comprising 20 different anti-sense DNAoligos, each of which is confined to a specific area in the array. Inother words, each of the 20 different anti-sense DNA oligonucleotidesare located in a small area of the array—the array therefore consists of20 areas each of which comprise antisense oligonucleotides of a givensequence.

Each of these 20 anti-sense DNA oligonucleotides comprise a sequence of10-15 nt that is complementary to the DNA label of one of the 20detection molecules.

Therefore, cells that are bound by a certain detection molecule willbecome immobilized to the array, through hybridization between the DNAoligonucleotide label of said certain detection molecule and theanti-sense DNA oligonucleotide of the array at that position. Thehybridization conditions may be adjusted so that hybridization betweenjust one detection molecule and one anti-sense DNA leads to inefficientimmobilization of the corresponding cell, whereas hybridization ofseveral detection molecules to several anti-sense DNA leads to efficientimmobilization of the corresponding cell.

As a result, the cells bound by a certain kind of detection molecules(i.e. bound to a certain specificity of detection molecule) will becomeimmobilized to one of the 20 areas of the array, defined by the sequenceof the detection molecule bound to the cell and the position in thearray of the antisense DNA oligonucleotide that is complementary to saiddetection molecule's label. Several cells, each bound by a large numberof detection molecules, may become immobilized in a certain area of thearray. Because of the fluorescent PE molecules attached to the detectionmolecule, this will give rise to a flouorescent signal from this area ofthe array. Moreover, at higher resolution (using a microscope) it willbe possible to count the number of cells immobilized in the specificarea of the array, and finally, the fluorescence of each cell andtherefore the relative number of detection molecules bound to each cell,may be determined. See the figure below showing an example where thecell sample comprises two cell specificities that are bound by detectionmolecules; one of the specificities are represented by 12 cells, theother is represented by 5 cells. In FIG. 17 , hatched areas representfluorescent light above a certain threshold.

In this example, the fluid cell sample could be of any kind (e.g.synovial fluid, blood, bone marrow, environmental sample (e.g.comprising bacterial cells, in which case the binding molecules could beantibodies binding to proteins of different bacterial surfaces), etc.

The number of different detection molecules could be expanded to muchlarger numbers, e.g. 100, 1.000 or 10.000.

Rather than having the detection molecules carry the fluorescentmolecules directly, a system of primary and secondary antibodies and acorresponding staining system could be used, like standard secondarylabelling systems, in order to increase the signal strength.

Example 80

In this example, the labels used are DNA oligonucleotides of differentlength. The identity of the individual label is based on its mass, byeither mass spectrometry or differential migration in a gelelectrophoresis analysis.

-   -   1. Sample preparation.

Blood comprising different kinds of blood cells is drawn from a person,and used directly in the incubation, step E below. Alternatively, anytype of sample described in the examples above can be used.

-   -   3. Binding molecules preparation

The binding molecules used in this study are 10 different antibodies,recognizing 10 different cell differentiation markers (CDs)respectively, namely CD3, CD4, CD8, CD16, CD56, . . . **. Each of theseare commercially available.

-   -   4. Label preparation

10 DNA oligonucleotides of length 50 nt, 100 nt, 150 nt, 200 nt, 250 nt,300 nt, 350 nt, 400 nt, 450 nt, and 500 nt, respectively, with aterminal N-succinimidyl ester moiety are prepared by standard means.

-   -   5. Detection molecules preparation

Each of the 10 antibodies mentioned under (B) are incubated inappropriate buffer (e.g. PBS pH 8) together with one of the 10oligonucleotides mentioned in step (C) above, allowing theN-succinimidyl ester to react with free amines on the surface of theantibody, to form a covalent link between the antibody and theoligonucleotide. In this way, 10 detection molecules are generated, eachof which comprise one specific antibody (e.g. anti-CD3 antibody) and onespecific oligonucleotide (e.g. 50 nt DNA oligo).

-   -   6. Incubation of sample and detection molecules

The sample of step (A) is incubated with the 10 detection moleculesunder appropriate conditions (e.g. appropriate buffer, e.g. Tris pH 7.5is added, or the oligonucleotides from step (D) in e.g. PBS pH 8 issimply mixed with sample without further addition of buffer. Incubationcan be at 0, 4, 10, 20, 30, or 40 degrees celcius.

-   -   7. Isolating bound and unbound detection molecules

The incubation mixture of step (E) is centrifuged, to recover all cellsof the sample. Supernatant is removed, the cells resuspended and bufferadded. Centrifugation is repeated 1-3 times, and the cells finallyresuspended. The suspension of cells will contain detection moleculesthat are capable of binding to one or more cells. Thus, the detectionmolecules of the suspension are representative of the distribution ofreceptors on the cell surface of the cells of the original sample.

-   -   8. Determining the identity and amount of the recovered        detection molecules.

1 μL of the mixture of cells and bound detection molecules aretransferred to a 100 μL PCR reaction comprising forward and reverseprimers that can anneal to each of the 10 oligonucleotide labelsdescribed in step (C) above, and comprising all other components for anefficient PCR reaction. The PCR reaction is performed under standardconditions, and aliquots are taken out after 25, 30 and 35 cycles areperformed.

The PCR product is applied to a gel capable of resolving the individualoligonucleotide fragments (e.g. a 2.5% agarose gel), and electrophoresisis performed. Once the fastest moving product (corresponding to the 50nt oligonucleotide) has migrated about ¾ of the gel length,electrophoresis is terminated. The position of a band in the gelreflects its size and therefore identifies the correspondingoligonucleotide label; the uppermost band represents the largestfragment (500 nt), and the lowermost band represents the smallestfragment (50 nt). The intensity of each of the 10 bands, correspondingto each of the 10 different oligonucleotide fragments, is indicative ofthe relative amount recovered of each oligonucleotide label andtherefore, of each detection molecule.

Example 81

This example is as example 80, except that the labels used here are PNAfragments rather than DNA oligonucleotides, and the identity of thelabels of the recovered detection molecules are determined by massspectrometry analysis rather than gel electrophoresis.

Sample and binding molecules are as described in example 80.

Labels are prepared as follows. 10 different PNAs are prepared, ofdifferent size (e.g. comprising 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23bases). During synthesis, N-succinimidyl ester is introduced at one ofthe PNA fragment. Further, also during its synthesis, a disulfide bond(S—S) is introduced between the ester and the rest of the PNA fragment.Thus, in the label a disulfide bond links the N-succinimidyl ester withthe remainder of the PNA fragment.

Detection molecules are prepared as in example 80, except that the PNAlabels are used instead of the DNA oligonucleotide labels of example 80.Each of the resulting 10 detection molecules therefore consists of aspecific binding molecule (a specific antibody, e.g. CD3) and a specificPNA label (e.g. PNA comprising 5 bases). Isolation of detectionmolecules capable of binding to cells of the sample is done bycentrifugation as described in example 80, and the final cell suspensionthus contains cells plus detection molecules capable of binding to cellsof the sample.

Finally, the identity and amount of the recovered detection molecules isdetermined in the following way: First, the PNA labels are cleaved offfrom the detection molecules (and therefore, released from the cellsthat the detection molecule is bound to) by addition of DTT, whichcleaves the disulfide bond. The cells are spun down by centrifugation,and the supernatant (comprising the released PNA labels but not thecells) is then subjected to a mass spectrometry analysis.

The mass spectrometry analysis will reveal the relative amount of eachof the 10 PNA labels (corresponding to the relative amount of each ofthe 10 detection molecules recovered by centrifugation of the cells).

Example 82

This example is as example 81, except that the labels used here arepeptide fragments rather than PNA fragments

Labels are prepared as follows. 10 different peptides are prepared, ofdifferent size (e.g. comprising 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23amino acids). During synthesis, N-succinimidyl ester is introduced atone of the peptide fragment. Further, also during its synthesis, adisulfide bond (S—S) is introduced between the ester and the rest of thepeptide fragment. Thus, in the label a disulfide bond links theN-succinimidyl ester with the remainder of the peptide fragment.

Detection molecules are prepared as in example 81, except that thepeptide labels are used instead of the PNA labels of example 81. Each ofthe resulting 10 detection molecules therefore consist of a specificbinding molecule (a specific antibody, e.g. CD3) and a specific peptidelabel (e.g. peptide comprising 5 amino acids).

The mass spectrometry analysis will reveal the relative amount of eachof the 10 peptide labels (corresponding to the relative amount of eachof the 10 detection molecules recovered by centrifugation of the cells).

Example 120

This is an example where the samples are three buffy coats (BC's), theLinker is PE and streptavidin conjugated dextran, and the BindingMolecules are biotinylated peptide-MHC (pMHC) complexes. The Labels aresynthetic oligonucleotides modified with a terminal biotin capture-tag.The labels are combined oligonucleotide label arising by annealing an Aoligonucleotide (modified with biotin) to a partially complimentary Boligonucleotide label followed by enzymatic DNA polymerase extension ofOligo A and Oligo B to create a fully double stranded label.

The Detection Molecules are created by combining biotinylated pMHC andlabels in the form of biotin-modified oligonucleotide onto astreptavidin-modified dextran linker. The detection molecule furthercontained a fluorochrome (PE). 30 different Detection Molecules aregenerated wherein the individual detection molecules containingdifferent pMHC are encoded by corresponding individual oligonucleotidelabels. Two versions (Set 1 and Set 2, see table x) of each of the 30different Detection Molecules are created in the way that all Detectionmolecules are generated in two forms with different labels. Threesamples, PBMC's, were incubated with an amount of mixed DetectionMolecules.

In this example CD8+ T cell-bound Detection Molecules are separated fromthe non-cell bound Detection Molecules by capture of CD8+ T cells usinganti CD8-coated magnetic Dynabeads. Magnetic bead-isolated CD8-positiveT cells are subjected to PCR amplification of associated Label and thePCR product is analyzed as a read of the oligonucleotide labelassociated with the detection molecules bound to the isolated cells thusrevealing the identity of detection molecules bound to the T cellspresent in the sample.

-   -   1. Sample preparation.        -   a. Acquiring sample: The cell sample used in this experiment            is obtained by preparing PBMC's from blood drawn from three            donors (D131, D149 and D158) that, by conventional            MHC-multimer staining, are characterized for their specific            T cells towards a number of virus antigens.        -   b. Modifying sample: The PBMC's are prepared as described in            example 1.    -   2. Linker preparation: The Linker is PE and streptavidin        conjugated dextran (Dextramer from Immudex) which is prepared as        in example 1.    -   3. Binding molecules preparation: Binding Molecules are        biotinylated pMHC complexes with peptide/HLA combination as        described in table below

Labels Labels set 1 set 2  HLA Antigen Peptide 2OS-A1-B2 2OS-A1-B12 A2CMV pp65 NLVPMVATV 2OS-A1-B3 2OS-A1-B13 A2 EBV-E3B LLDFVRFMGV 2OS-Al-B42OS-A1-B14 A2 HPV E6 TIHDIILECV 2OS-Ai-B5 2OS-A1-B15 A2 HSV-1gBRMLGDVMAV 2OS-Al-B6 2OS-A1-B16 A2 Neg Control ALIAPVHAV 2OS-A2-B22OS-A2-B12 A2 CMV-IE1 VLEETSVML 2OS-A2-B3 2OS-A2-B13 A2 EBV-BALF4FLDKGTYTL 2OS-A2-B4 2OS-A2-B14 A2 FLU GILGFVFTL 2OS-A2-B5 2OS-A2-B15 A2MART-1 ELAGIGILTV 2OS-A2-B6 2OS-A2-B16 A3 Cancer-gp100 ALLAVGATK2OS-A3-B2 2OS-A3-B12 A2 EBV-EBMAC3 CLGGLLTMV 2OS-A3-B3 2OS-A3-B13 A2EBV-BMRF1 TLDYKPLSV 2OS-A3-B4 2OS-A3-B14 A2 FLU-BNP KLGEFYNQMM 2OS-A3-B52OS-A3-B15 A2 HIV-gag SLYNTVATL 2OS-A3-B6 2OS-A3-B16 B07 Neg ControlGPAESAAGL 2OS-A4-B2 2OS-A4-B12 A2 EBV-BMLF1 GLCTLVAML 2OS-A4-B32OS-A4-B13 A2 EBV-BRLF1 YVLDHLIVV 2OS-A4-B4 2OS-A4-B14 A2 HSV1gBGIFEDRAPV 2OS-A4-B5 2OS-A4-B15 A2 HIV ILKEPVHGV 2OS-A4-B6 2OS-A4-B16 B08Neg Control AAKGRGAAL 2OS-A5-B2 2OS-A5-B12 A2 EBV-LMP1 YLLEMLWRL2OS-A5-B3 2OS-A5-B13 A2 HPV-E7 YMLDLQPETT 2OS-A5-B4 2OS-A5-B14 A2 Hsv1gBYLANGGFLI 2OS-A5-B5 2OS-A5-B15 A2 HIV FLGKIWPS 2OS-A5-B6 2OS-A5-B16 H2KbNeg Control AYAGSAGSI 2OS-A6-B2 2OS-A6-B12 A2 EBV-LMP1 YLQQNVWVTL2OS-A6-B3 2OS-A6-B13 A2 HPV-E7 LLMGTLGIVC 2OS-A6-B4 2OS-A6-B14 A2HSV-U125 FLWEDQTLL 2OS-A6-B5 2OS-A6-B15 A1 Neg Control STEGGGLAY2OS-A6-B6 2OS-A6-B16 Neg Control no peptide

-   -   4. Label preparation: Label is generated as described in example        4 except that in this example only 2×60 labels are used (See        table above)    -   5. Detection molecules preparation        -   a. Synthesis            -   i. According to table below transfer the required volume                Dextran conjugate to an Eppendorf tube. Centrifuge                (10.000 g, 10 min, 4° C.).            -   ii. Transfer the supernatant containing dextrane                conjugate without precipitates to 2*30 wells on two                plates.            -   iii. In a 96 well format add the 2×30 Label oligos in                2:1 molar concentration to PE-Dextramer conjugate.            -   iv. Mix well and incubate 30 min, 4° C.            -   v. Thaw pMHC binding molecules on ice, dilute to 100                μg/mL in PBS and centrifuge (2000 g, 5 minutes, 18° C.).            -   vi. Transfer the pMHC monomers to wells of the 96 well                plate according to the above table to allow the                combination of pMHC with labeled linker.            -   vii. Mix well and incubate 30 minutes at r.t. and PBS                subsequently.

Detection molecule preparation Concentration Dextran conjugate 16*10{circumflex over ( )}-8M Oligo lable 54.25 *10{circumflex over( )}-8M Detection molecules/samples Number of Detection molecules 30Number of samples 20 Assembling Label and Linker Volumes needed 1:Transfer Dextran to Eppendorf 630 μl tube and centrifuge 10000 g, 5 min,4 C. 2: Transfer the supernatant to wells 20 μl/well on a plateaccording to setup 3: Add oligo labels to wells according 11.8 μl/wellto setup, Mix and incubate 30 min, 4 C. Preparation of bindingmolecules: 1: Thaw pMHC monomers on ice 2: Dilute monomers in PBS to 100μg/ml = 2 μM Vtotal needed per pMHC per plate 40 μl/pMHC Preparation ofDetection Molecules 1: Centrifuge pMHC monomers 2000 g, 10 min 18 C. 2:Transfer supernatant to the 96well 26.4 μl/well plate (To theDextran-MHC control is an equal volume of PBS added) 3: Mix well andincubate 30 min at RT 4: Add PBS 1.8 μl/well 5: Store at 4 C. Vtotal ineach well 60.0

-   -   -   a. Modification: No further        -   b. Purification: No further

    -   6. Incubation of sample and detection molecules        -   a. Amount of sample: 2 million PBMC's per sample        -   b. Amount of detection molecule: According to table below.            Each donor sample is incubated with 4 different amounts of            Detection Molecule        -   c. Conditions:            -   i. PBMC's are thawed in 10 ml RPM1-10% FCS and washed                twice in 2 mL RPMI-10% FCS. Cells are then washed in 2                mL PBS w. 0.5% BSA, 100 μg/ml Herring DNA, 2 mM EDTA.                (All washing of cells in this experiment refers to                centrifuge for 5 min at 800 g to collect cells followed                by discarding of wash buffer)            -   ii. Resuspend cells in 500 μl PBS w. 0.5% BSA, 100 μg/ml                Herring DNA, 2 mM EDTA to a concentration 20 million/ml.            -   iii. Add 25 μl 1 μM Dasatinib and incubate 30 min at                37° C. (Final Dasatinib conc. 50 nM).            -   iv. Centrifuge Detection Molecules (2000 g, 5 minutes,                18° C.)            -   v. Add 166.5 μl 10 μM biotin to an Eppendorf tube.            -   vi. Pool 25 μl of each Detection Molecule from each of                the two sets into the Eppendorf tube (Detection Molecule                library) Vtot=1666, 5 μl which is enough for 3 donors in                4 concentrations.            -   vii. Centrifuge the library of Detection Molecules                (10000 g, 10 min) and transfer 1550 μl to a new tube                avoiding aggregates.            -   viii. Transfer from new tube to 5 ml tubes according to                setup/volumes below.            -   ix. To all twelve tubes add 100 μl cells (2 million                cells) of the respective donors, to a VTot in each                sample of 300 μl. Incubate 15 minutes at 37° C.            -   x. Wash cells twice in 2 ml PBS w. 0.5% BSA, 100 μg/ml                Herring DNA, 2 mM EDTA (Centrifuge 5 min at 800 g)

3 μl 1.5 μl 0.75 μl 0.375 μl Sample/Detect Mol D131 S1: 200 μl S2: 100μl S3: 50 μl S4: 25 μl D149 S5: 200 μl S6: 100 μl S7: 50 μl S8: 25 μlD158 S9: 200 μl S10: 100 μl S11: 50 μl S12: 25 μl PBS w. 0.5% BSA, 100μg/ml Herring DNA, 2 mM EDTA D131 S1: 0 μl S2: 100 μl S3: 150 μl S4: 175μl D149 S5: 0 μl S6: 100 μl S7: 150 μl S8: 175 μl D158 S9: 0 μl S10: 100μl S11: 150 μl S12: 175 μl

-   -   7. Enrichment of detection molecules with desired        characteristics        -   a. Apply: In this example Detection Molecules are isolated            by capturing CD8+ cells using anti CD8 antibody coated            magnetic beads (DYNABEADS CD8, #11147D, Life Technologies).            Detection molecules associated with captured CD8+ cells are            isolated. Dynabeads used according to manufacturer's            protocol.            -   i. Briefly; Dynabeads are vortex >30 sec and transfer 50                μl/sample=600 μl to a 5 ml tube and add 1 ml PBS+0.1%                BSA+2 mM EDTA. Re-suspended dynabeads are placed in                magnet 1 min, supernatant is discarded and dynabeads are                re-suspended in 600 μl PBS+0.1% BSA+2 mM EDTA.            -   ii. To all samples add 50 μl washed beads+950 μl                Isolation buffer=>Vtot=1 ml. Incubate 20 min 4 C on a                tilting plate.        -   b. Wash:            -   i. Place tubes in magnet (Dynal, Life Technologies) for                2 min and carefully remove supernatant            -   ii. Wash twice: Add 2 ml PBS+0.1% BSA+2 mM EDTA, vortex,                place in magnet 2 min, remove supernatant.            -   iii. Re-suspend beads in 500 μl PBS w. 0.5% BSA, 100                μg/ml Herring DNA, 2 mM EDTA, transfer to Eppendorf                tubes.            -   iv. Centrifuge tubes 5 min at 5000 g, remove supernatant                final volume app. 20 μl→Store at −80 C or store on ice                at 4 C O.N for PCR        -   c. Separate: Is done during washing.    -   8. Identification of enriched detection molecule: Separated        Detection Molecules are analyzed by PCR amplification of the        attached labels followed by sequencing of the PCR products to        reveal the identity of isolated detection molecules and thus the        identity of those antigens for which specific T cells were        present in the sample. The PCR amplification and sequencing of        PCR product is done as for example 3 using the same sets of        primers and the same sequencing service and sequencing        de-convolution service.

Example 121

This is an example where the samples are PBMC's prepared as three buffycoats (BC's), the Linker is PE and streptavidin conjugated dextran, andthe Binding Molecules are biotinylated peptide-MHC (pMHC) complexes. TheLabels are synthetic oligonucleotides modified with a terminal biotincapture-tag.

In this example CD8+ T cell-bound Detection Molecules are separated fromnon-cell bound Detection Molecules by capture of CD8+ T cells bymagnetic labeling of CD8+ cells with CD8 MicroBeads (CD8 MicroBeads,human, #130-045-201, Miltenyi, Germany). Magnetic bead-isolatedCD8-positive T cells are subjected to PCR amplification of associatedLabel (as described in example 3) and the PCR product is analyzed as aread of the oligonucleotide label associated with the detectionmolecules bound to the isolated cells thus revealing the identity ofdetection molecules bound to the T cells present in the sample (asexample 3).

-   -   1. Sample preparation. The samples are PBMC's from three donor        materials prepared as described in example 3.    -   2. Linker preparation: The Linker is PE and streptavidin        conjugated dextran (Dextramer from Immudex) which is prepared as        in example 1.    -   3. Binding molecules preparation: Binding Molecules are        biotinylated pMHC complexes with peptide/HLA combination as        described in example 3.    -   4. Label preparation: Labels are biotin modified DNA        oligonucleotides as described in example 3.    -   5. Detection molecules preparation: Detection Molecules are        prepared by combining 110 aliquots of Linker with individual        Labels followed by adding pMHC Binding Molecules as described in        Example 3.    -   6. Incubation of sample and detection molecules: PBMC's are        mixed with the 110 member library of Detection Molecules as        described in example 3.    -   7. Enrichment of detection molecules with desired        characteristics:        -   a. Apply: In this example Detection Molecules are isolated            by capturing CD8+ cells using anti CD8 antibody coated            magnetic beads (CD8 MicroBeads, human, #130-045-201,            Miltenyi, Germany). Detection molecules associated with            captured CD8+ cells are isolated. CD8 MicroBeads are used            according to manufacturer's protocol. Briefly;            -   i. Centrifuge cell suspensions at 300×g for 10 minutes.                Aspirate supernatant completely.            -   ii. Resuspend cell pellets in 80 μL of buffer                (phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine                serum albumin (BSA), and 2 mM EDTA).            -   iii. Add 20 μL of CD8 MicroBeads. Mix well and incubate                for 15 minutes in the refrigerator (2-8)            -   iv. Wash cells by adding 1-2 mL of buffer and centrifuge                at 300×g for 10 minutes. Aspirate supernatant                completely. Resuspend cells in 500 μL of buffer.            -   v. Place an MS column (Miltenyi, Germany) in the                magnetic field of a OctoMACS column stand Separator.                Prepare column by rinsing with 500 μL of buffer:            -   vi. Apply cell suspension onto the column. Collect                unlabeled cells that pass through and wash column with                500 μL of buffer. Collect total effluent; this is the                unlabeled cell fraction. Perform washing steps by adding                buffer three times. Only add new buffer when the column                reservoir is empty.            -   vii. Remove column from the separator and place it on a                suitable collection tube.            -   viii. Pipette 1 mL of buffer onto the column.            -   ix. Immediately flush out the magnetically labeled cells                by firmly pushing the plunger into the column.            -   x. Centrifuge tubes 5 min at 500 g, remove supernatant.                The final volume of app. 20 μl containing collected CD8+                cells and their associated Detection Molecules are store                at −80 C for later analysis.    -   8. Identification of enriched detection molecule: Separated        cells and their associated Detection Molecules are analyzed by        PCR amplification of the attached labels followed by sequencing        of the PCR products to reveal the identity of isolated Detection        Molecules and thus the identity of those antigens for which        specific T cells were present in the sample. The identification        is done as for example 3.

Example 122

This is an example where the samples are PBMC's prepared as three buffycoats (BC's), the Linker is PE and streptavidin conjugated dextran, andthe Binding Molecules are biotinylated peptide-MHC (pMHC) complexes. TheLabels are synthetic oligonucleotides modified with a terminal biotincapture-tag.

In this example, though, all cells and their bound Detection Moleculesare first separated, by centrifugation, from the supernatant containingDetection Molecules not bound to cells. Secondly, remaining DetectionMolecules are captured by their PE modification on the Linker using antiPE MicroBeads (anti PE MicroBeads, #130-048-801, Miltenyi, Germany).Magnetic bead-isolated cells are subjected to PCR amplification ofDetection-Molecule associated Labels (as described in example 3) and thePCR product is analyzed as a read of the oligonucleotide labelassociated with the detection molecules bound to the isolated cells thusrevealing the identity of detection molecules bound to the T cellspresent in the sample as in example 3.

-   -   1. Sample preparation. The samples are PBMC's from three donor        materials prepared as described in example 3.    -   2. Linker preparation: The Linker is PE and streptavidin        conjugated dextran (Dextramer from Immudex) which is prepared as        in example 1.    -   3. Binding molecules preparation: Binding Molecules are        biotinylated pMHC complexes with peptide/HLA combination as        described in example 3.    -   4. Label preparation: Labels are biotin modified DNA        oligonucleotides as described in example 3.    -   5. Detection molecules preparation: Detection Molecules are        prepared by combining 110 aliquots of Linker with individual        Labels followed by adding pMHC Binding Molecules as described in        Example 3.    -   6. Incubation of sample and detection molecules: PBMC's are        mixed with the 110 member library of Detection Molecules as        described in example 3.    -   7. Enrichment of detection molecules with desired        characteristics        -   a. Apply: In this example Detection Molecules are isolated            by centrifugation separation of cells and their bound            Detection Molecules from the supernatant containing            Detection Molecules not bound to cells followed by magnetic            capture of cells with bound PE-labeled Detection Molecules            thereby separating from cells without bound PE-labeled            Detection Molecules using anti PE MicroBeads (anti PE            MicroBeads, #130-048-801, Miltenyi, Germany). MicroBeads are            used according to manufacturer's protocol. Briefly;            -   i. Centrifuge cell suspensions at 300×g for 10 minutes.                Aspirate supernatant completely.            -   ii. Wash cells twice by adding 2 mL of buffer                (phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine                serum albumin (BSA), and 2 mM EDTA) and centrifuge at                300×g for 10 minutes. Aspirate supernatant completely.            -   iii. Resuspend cell pellets in 80 μL of buffer.            -   iv. Add 20 μL of anti PE MicroBeads. Mix well and                incubate for 15 minutes in the refrigerator (2-8)            -   v. Wash cells by adding 1-2 mL of buffer and centrifuge                at 300×g for 10 minutes. Aspirate supernatant                completely. Resuspend cells in 500 μL of buffer.            -   vi. Place an MS column (Miltenyi, Germany) in the                magnetic field of a OctoMACS column stand Separator.                Prepare column by rinsing with 500 μL of buffer:            -   vii. Apply cell suspension onto the column. Collect                unlabeled cells that pass through and wash column with                500 μL of buffer. Collect total effluent; this is the                unlabeled cell fraction. Perform washing steps by adding                buffer three times. Only add new buffer when the column                reservoir is empty.            -   viii. Remove column from the separator and place it on a                suitable collection tube.            -   ix. Pipette 1 mL of buffer onto the column.            -   x. Immediately flush out the magnetically labeled cells                by firmly pushing the plunger into the column.            -   xi. Centrifuge tubes 5 min at 500 g, remove supernatant.                The final volume of app. 20 μl containing collected CD8+                cells and their associated Detection Molecules are store                at −80 C for later analysis.    -   8. Identification of enriched detection molecule: Separated        cells and their associated Detection Molecules are analyzed by        PCR amplification of the attached labels followed by sequencing        of the PCR products to reveal the identity of isolated Detection        Molecules and thus the identity of those antigens for which        specific T cells were present in the sample. The identification        is done as for e.g. example 6.

Example 123

This is an example where the samples are PBMC's prepared as three buffycoats (BC's), the Linker is PE and streptavidin conjugated dextran, andthe Binding Molecules are biotinylated peptide-MHC (pMHC) complexes. TheLabels are synthetic oligonucleotides modified with a terminal biotincapture-tag.

In this example, cells and their bound Detection Molecules areseparated, by centrifugation, from the supernatant containing DetectionMolecules not bound to cells. The DNA oligonucleotide Labels onDetection Molecules associated with captured cells are subjected to PCRamplification (as example 3) and the PCR product is analyzed as a readof the oligonucleotide label associated with the detection moleculesbound to the isolated cells thus revealing the identity of detectionmolecules bound to the T cells present in the sample (as example 3).

-   -   1. Sample preparation. The samples are PBMC's from three donor        materials prepared as described in example 3.    -   2. Linker preparation: The Linker is PE and streptavidin        conjugated dextran (Dextramer from Immudex) which is prepared as        in example 1.    -   3. Binding molecules preparation: Binding Molecules are        biotinylated pMHC complexes with peptide/HLA combination as        described in example 3.    -   4. Label preparation: Labels are biotin modified DNA        oligonucleotides as described in example 3.    -   5. Detection molecules preparation: Detection Molecules are        prepared by combining 110 aliquots of Linker with individual        Labels followed by adding pMHC Binding Molecules as described in        Example 3.    -   6. Incubation of sample and detection molecules: PBMC's are        mixed with the 110 member library of Detection Molecules as        described in example 3.    -   7. Enrichment of detection molecules with desired        characteristics        -   b. Apply: Detection Molecules are isolated by            centrifugation-separation of cells and their bound Detection            Molecules from the supernatant containing Detection            Molecules not bound to cells. Briefly;            -   i. Centrifuge cell suspensions at 300×g for 10 minutes.                Aspirate supernatant completely.            -   ii. Wash cells twice by adding 2 mL of buffer                (phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine                serum albumin (BSA), and 2 mM EDTA) and centrifuge at                300×g for 10 minutes.            -   iii. Aspirate supernatant completely. The final volume                of app. 20 μl containing collected cells and their                associated Detection Molecules are store at −80C for                later analysis.    -   8. Identification of enriched Detection Molecule: Separated        cells and their associated Detection Molecules are analyzed by        PCR amplification of the attached labels followed by sequencing        of the PCR products to reveal the identity of isolated Detection        Molecules and thus the identity of those antigens for which        specific T cells were present in the sample. The identification        is done as for example 3.

Example 124

This is an example where the samples are PBMC's prepared as three buffycoats (BC's), the Linker is PE and streptavidin conjugated dextran, andthe Binding Molecules are biotinylated peptide-MHC (pMHC) complexes. TheLabels are synthetic oligonucleotides modified with a terminal biotincapture-tag.

In this example, INFγ producing cells and their bound DetectionMolecules are separated from Detection Molecules not bound to INFγproducing cells by magnetic labeling and capture of INFγ producing cellsusing the MicroBead based INFγ Secretion Assay (INFγ Secretion Assay,#130-054-201, Miltenyi, Germany). Magnetic bead-isolated INFg producingcells are subjected to PCR amplification of associated DNAoligonucleotide Label (as described in example 3) and the PCR product isanalyzed as a read of the oligonucleotide label associated with thedetection molecules bound to the isolated cells thus revealing theidentity of detection molecules bound to the INFγ producing cellspresent in the sample (as example 3).

-   -   1. Sample preparation. The samples are PBMC's from three donor        materials prepared as described in example 3.    -   2. Linker preparation: The Linker is PE and streptavidin        conjugated dextran (Dextramer from Immudex) which is prepared as        in example 1.    -   3. Binding molecules preparation: Binding Molecules are        biotinylated pMHC complexes with peptide/HLA combination as        described in example 3.    -   4. Label preparation: Labels are biotin modified DNA        oligonucleotides as described in example 3.    -   5. Detection molecules preparation: Detection Molecules are        prepared by combining 110 aliquots of Linker with individual        Labels followed by adding pMHC Binding Molecules as described in        Example 3.    -   6. Incubation of sample and detection molecules: PBMC's are        mixed with the 110 member library of Detection Molecules as        described in example 3.    -   7. Enrichment of detection molecules with desired        characteristics        -   i. Apply: In this example Detection Molecules are isolated            by capturing INF γ producing cells using anti INF γ catch            antibody in combination with anti INF γ detection antibody.            All reagents and procedures as described by Miltenyi (INF γ            Secretion Assay, #130-054-201, Miltenyi, Germany). Finally            INFγ producing cells are captured using anti PE Microbeads.        -   ii. Centrifuge tubes 5 min at 500 g, remove supernatant. The            final volume of app. 20 μl containing collected INFγ            producing cells and their associated Detection Molecules are            store at −80 C for later analysis.    -   8. Identification of enriched detection molecule: Separated        cells and their associated Detection Molecules are analyzed by        PCR amplification of the attached labels followed by sequencing        of the PCR products to reveal the identity of isolated Detection        Molecules and thus the identity of those antigens for which        specific T cells were present in the sample. The identification        is done as for example 3.

Items Set #1

-   1. A method comprising the following steps:    -   a. Combining at least one cell with at least one detection        molecule, where the detection molecule comprises a binding        molecule (BM), a linker (Li), and a label (La);    -   b. Allowing the detection molecules to recognize and bind cells        through their binding molecule entity;    -   c. Detecting or isolating pairs of cell-detection molecule        complexes formed in step (b);    -   d. Identifying detection molecules capable of binding to a cell        in step (b).-   2. The method of item 1 where the binding molecule (BM) is a    peptide-MHC complex, an antibody or an oligonucleotide.-   3. The method of item 1 or 2 where the label (La) is an antibody, a    nucleic acid, a particle comprising an electronic or electromagnetic    signal, or a particle comprising a radio signal.-   4. The method of any of items 1-3 where the linker (Li) is a    streptavidin, polysaccharide, dextran, peptide, or carbon-based    polymer.-   5. The method of any of items 1-4 where step c is carried out by    immobilization of the cell-detection molecule pairs-   6. The method of item 5 where said immobilization is by    precipitating cells by centrifugation, immunoprecipitation of the    cells optionally involving centrifugation, or any other means that    precipitates the cells, leading to co-precipitation of detection    molecules bound to cells.-   7. The method of item 6 where said immobilization is by binding one    or more cells to a bead, particle or surface, or any other means    that immobilizes said one or more cells, leading to    co-immobilization of the detection molecules bound to said one or    more cells.-   8. The method of any of items 5-7 where said immobilization involves    the use of an antibody or other molecule, capable of specifically    binding a subset of cells-   9. The method of item 8 where said antibody or other molecule    specifically recognizes the T cell receptor (TCR) or the CD4 or CD8    proteins of T cells.-   10. The method of any of the preceding items where a label uniquely    identifies individual detection molecules or identifies specific    subsets of detection molecules-   11. The method of any of the preceding items where the Label is an    oligonucleotide.-   12. The method of any of the preceding items where the binding    molecule is a pMHC complex, an antibody or an oligonucleotide    aptamer.-   13. The method of any of the preceding items where the linker is a    dextran molecule, polysaccharide, oligonucleotide, streptavidin,    peptide, carbon-based molecule, carbohydrate or an organic molecule.-   14. A multimeric major histocompatibility complex (MHC) comprising    -   a. two or more MHC's linked by a backbone molecule; and    -   b. at least one nucleic acid molecule linked to said backbone,        wherein said nucleic acid molecule comprises a central stretch        of nucleic acids (barcode region) designed to be amplified by        e.g. PCR.-   15. The multimeric major histocompatibility complex according to    item 14, wherein the backbone molecule is selected from the group    consisting of polysaccharides, such as glucans such as dextran, a    streptavidin or a streptavidin multimer.-   16. The multimeric major histocompatibility complex according to    item 14 or 15, wherein the MHC's are coupled to the backbone through    a streptavidin-biotin binding, streptavidin-avidin.-   17. The multimeric major histocompatibility complex according to any    of the preceding items, wherein the MHC's are linked to the backbone    via the MHC heavy chain.-   18. The multimeric major histocompatibility complex (MHC) according    to any of the preceding items, wherein the MHC is artificially    assembled.-   19. The multimeric major histocompatibility complex (MHC) according    to any of the preceding items, composed of at least four MHC's, such    as at least eight, such as at least ten, 2-30, 2-20, such as 2-10 or    such as 4-10 MHC's.-   20. The multimeric major histocompatibility complex (MHC) according    to any of the preceding items, wherein the at least one nucleic acid    molecule is composed of at least a 5′ first primer region, a central    region (barcode region), and a 3′ second primer region.-   21. The multimeric major histocompatibility complex (MHC) according    to any of the preceding items, wherein the at least one nucleic acid    molecule has a length in the range 20-100 nucleotides, such as    30-100, such as 30-80, such as 30-50 nucleotides.-   22. The multimeric major histocompatibility complex (MHC) according    to any of the preceding items, wherein the at least one nucleic acid    molecule is linked to said backbone via a streptavidin-biotin    binding and/or streptavidin-avidin binding.-   23. The multimeric major histocompatibility complex (MHC) according    to any of the preceding items, wherein the at least one nucleic acid    molecule comprises or consists of DNA, RNA, and/or artificial    nucleotides such as PLA or LNA.-   24. The multimeric major histocompatibility complex (MHC) according    to any of the preceding items, wherein the MHC is selected from the    group consisting of class I MHC, a class II MHC, a CD1, or a    MHC-like molecule.-   25. The multimeric major histocompatibility complex (MHC) according    to any of the preceding items, wherein the backbone further    comprises one or more linked fluorescent labels.-   26. A composition comprising a subset of multimeric major    histocompatibility complexes (MHC's) according to any of items    14-25, wherein each set of MHC's has a different peptide decisive    for T cell recognition and a unique “barcode” region in the DNA    molecule.-   27. The composition according to item 26, wherein the primer regions    in the DNA molecule are identical for each set of MHC's.-   28. A) The composition according to item 26 or 27, comprising at    least 10 different sets of MHC's such as at least 100, such as at    least 500, at least 1000, at least 5000, such as in the range    10-50000, such as 10-1000 or such as 50-500 sets of MHC's.-   28. B) A kit of parts comprising    -   a. a composition according to any of items 26 to 28; and    -   b. one or more sets of primers for amplifying the nucleic acid        molecules.-   29. A method for detecting antigen responsive cells in a sample    comprising:-   i) providing one or more multimeric major histocompatibility    complexes (MHC's) according to any of items 1-12 or a composition    according to any of items 14-16; ii) contacting said multimeric    MHC's with said sample; and detecting binding of the multimeric    MHC's to said antigen responsive cells, thereby detecting cells    responsive to an antigen present in a set of MHC's, wherein said    binding is detected by amplifying the barcode region of said nucleic    acid molecule linked to the one or more MHC's.-   30. The method according to item 29, wherein unbound MHC's are    removed before amplification, e.g. by washing and/or spinning.-   31. The method according to item 29 or 30, wherein the sample is a    blood sample, such as an peripheral blood sample, a blood derived    sample, a tissue biopsy or another body fluid, such as spinal fluid,    or saliva.-   32. The method according to any of items 29-31, wherein said sample    has been obtained from a mammal, such as a human, mouse, pigs,    and/or horses.-   33. The method according to any of item 30-32, wherein the method    further comprises cell sorting by e.g. flow cytometry such as FACS.-   34. The method according to any of items 29-33, wherein said binding    detection includes comparing measured values to a reference level,    e.g. a negative control and/or total level of response.-   35. The method according to any of item 29-34, wherein said    amplification is PCR such as QPCR.-   36. The method according to any of items 29-35, wherein the    detection of barcode regions includes sequencing of said region such    as deep sequencing or next generation sequencing.-   37. Use of a multimeric major histocompatibility complex (MHC)    according to any of items 14-25 or a composition according to any of    items 26-29 for the detecting of antigen responsive cells in a    sample.-   38. Use of a multimeric major histocompatibility complex (MHC)    according to any of items 14-25 or a composition according to any of    items 26-29 in the diagnosis of diseases or conditions, preferably    cancer and/or infectious diseases.-   39. Use of a multimeric major histocompatibility complex (MHC)    according to any of items 14-25 or a composition according to any of    items 26-29 in the development of immune-therapeutics.-   40. Use of a multimeric major histocompatibility complex (MHC)    according to any of items 14-25 or a composition according to any of    items 26-29 in the development of vaccines.-   41. Use of a multimeric major histocompatibility complex (MHC)    according to any of items 14-25 or a composition according to any of    items 26-29 for the identification of epitopes.    References-   1. Altman J D, Moss P A, Goulder P J, Barouch D H, McHeyzer-Williams    M G, Bell J I, et al. Phenotypic analysis of antigen-specific T    lymphocytes. Science. 1996; 274:94-6.-   2. Davis M M, Bjorkman P J. T-cell antigen receptor genes and T-cell    recognition. Nature. 1988; 334:395-402.-   3. Robins H S, Campregher P V, Srivastava S K, Wacher A, Turtle C J,    Kahsai O, et al. Comprehensive assessment of T-cell receptor    beta-chain diversity in alphabeta T cells. Blood. 2009;    114:4099-107.-   4. Hadrup S R, Bakker A H, Shu C J, Andersen R S, van V J, Hombrink    P, et al. Parallel detection of antigen-specific T-cell responses by    multidimensional encoding of MHC multimers. Nature Methods. 2009;    6:520-6.-   5. Andersen R S, Kvistborg P, March T F, Pedersen N W, Lyngaa R,    Bakker A H, et al. Parallel detection of antigen-specific T-cell    responses by combinatorial encoding of MHC multimers. NatProtoc.    2012-   6. Newell E W, Sigal N, Nair N, Kidd B a, Greenberg H B, Davis M M.    Combinatorial tetramer staining and mass cytometry analysis    facilitate T-cell epitope mapping and characterization. Nat    Biotechnol. 2013; 1-9.-   7. Soen Y, Chen D S, Kraft D L, Davis M M, Brown P O. Detection and    characterization of cellular immune responses using peptide-MHC    microarrays. PLoSBiol. 2003; 1:429-38.-   8. Stone J D, Demkowicz Jr. W E, Stern L J. HLA-restricted epitope    identification and detection of functional T cell responses by using    MHC-peptide and costimulatory microarrays. Proc Natl Acad Sci USA.    2005; 102:3744-9.-   9. Newell E W, Davis M M. Beyond model antigens: high-dimensional    methods for the analysis of antigen-specific T cells. Nat    Biotechnol. 2014; 32.-   10. Dössinger G, Bunse M, Bet J, Albrecht J, Paszkiewicz P J,    Weiβbrich B, et al. MHC multimer-guided and cell culture-independent    isolation of functional T cell receptors from single cells    facilitates TCR identification for immunotherapy. PLoS One. 2013;    8:e61384.-   11. Cha E, Klinger M, Hou Y, Cummings C, Ribas A, Faham M, et al.    Improved Survival with T Cell Clonotype Stability After Anti-CTLA-4    Treatment in Cancer Patients. Sci Transl Med. 2014; 6:238ra70.-   12. Robert L, Tsoi J, Wang X, Emerson R O, Homet B, Chodon T, et al.    CTLA4 blockade broadens the peripheral T cell receptor repertoire.    Clin Cancer Res. 2014-   13. Morgan R A, Dudley M E, Wunderlich J R, Hughes M S, Yang J C,    Sherry R M, et al. Cancer Regression in Patients After Transfer of    Genetically Engineered Lymphocytes. Science. 2006.-   14. Pannetier C, Even J, Kourilsky P. T-cell repertoire diversity    and clonal expansions in normal and clinical samples. ImmunolToday.    1995; 16:176-81.-   15. Cameron B J, Gerry A B, Dukes J, Harper J V, Kannan V, Bianchi F    C, et al. Identification of a Titin-derived HLA-A1-presented peptide    as a cross-reactive target for engineered MAGE A3-directed T cells.    Sci Transl Med. 2013; 5:197ra103.-   16. Linette G P, Stadtmauer E a, Maus M V, Rapoport A P, Levine B L,    Emery L, et al. Cardiovascular toxicity and titin cross-reactivity    of affinity-enhanced T cells in myeloma and melanoma. Blood. 2013;    122:863-71.

Items Set #2

-   1. A detection molecule comprising    -   a. at least one binding molecule (BM),    -   b. at least one linker (Li), and    -   c. at least one label (La).-   2. A cell-detection molecule complex comprising    -   a. At least one detection molecule comprising a binding molecule        (BM), a linker (Li) and a label (La), and    -   b. at least one cell.-   3. A composition comprising two or more different detection    molecules, or two or more sets of different detection molecules,    each detection molecule comprising at least one binding molecule    (BM), at least one linker (Li) and at least one label (La),    -   wherein each of the two or more detection molecules, or two or        more sets of detection molecules, comprises a label which is        unique to and specific for the binding molecule of each of said        two or more different detection molecules.-   4. The composition according to item 3, said composition comprising    2 to 1,000,000 different detection molecules, such as 2, 3, 4, 5, 6,    7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,    25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40    different detection molecules or sets of different detection    molecules; for example 1-3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-30,    30-35, 35-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110,    110-120, 120-130, 130-140, 140-150, 150-175, 175-200, 200-250,    250-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900,    900-1000, 1000-1500, 1500-2000, 2000-3000, 3000-4000, 4000-5000,    5000-7500, 7500-10,000, 10,000-20,000, 20,000-50,000,    50,000-100,000, 100,000-200,000, 200,000-500,000, 500,000-1,000,000    different detection molecules, or sets of different detection    molecules.-   5. The detection molecule according to any of the preceding items,    wherein said binding molecule is capable of specifically associating    with, recognizing and/or binding to a structure belonging to or    associated with an entity in a sample, such as a target structure.-   6. The detection molecule according to any of the preceding items,    wherein said binding molecule associates with, recognizes and/or    binds to a marker molecule specific for a given cell or cell type.-   7. The detection molecule according to any of the preceding items,    wherein said binding molecule is capable of specifically associating    with, recognizing and/or binding to a specific cell type, such as    selected from the group consisting of immune cells, lymphocytes,    monocytes, dendritic cells, T-cells, B-cells, NK cells, CD4+ T    cells, CD8+ T cells, αβ T cells, invariant γδ T cells,    antigen-specific T-cells, cells comprising TCRs, cells comprising    BCRs, a specific cancer cell-   8. The detection molecule according to any of the preceding items,    wherein said binding molecule is capable of specifically associating    with, recognizing and/or binding to a target specifically associated    with an organ selected from the group consisting of lymph nodes,    kidney, liver, skin, brain, heart, muscles, bone marrow, skin,    skeleton, lungs, the respiratory tract, spleen, thymus, pancreas,    exocrine glands, bladder, endocrine glands, reproduction organs    including the phallopian tubes, eye, ear, vascular system, the    gastroinstestinal tract including small intestines, colon, rectum,    canalis analis and prostate gland.-   9. The detection molecule according to any of the preceding items,    wherein said binding molecule is peptide-based or protein-based.-   10. The detection molecule according to any of the preceding items,    wherein said binding molecule is an anti-target molecule capable of    specifically associating with, recognizing and/or binding to a    predetermined target structure.-   11. The detection molecule according to any of the preceding items,    wherein said binding molecule is an anti-target-molecule.-   12. The detection molecule according to any of the preceding items,    wherein said binding molecule is selected from the group consisting    of an antibody, an antibody mimetic, an antibody-like molecule, a    peptide, an oligonucleotide, a peptide aptamer, a nucleic acid    aptamer, a DNA aptamer, an RNA aptamer, an XNA aptamer, a ligand, a    natural ligand, a variant or fragment of a natural ligands, a    synthetic ligand and a small organic molecule.-   13. The detection molecule according to any of the preceding items,    wherein said antibody mimetic is selected from the group consisting    of affibody molecules, affilinns, affimers, affitins, alphabodies,    anticalins, avimers, DARPins, fynomers, Kunitz domain peptides and    monobodies.-   14. The detection molecule according to any of the preceding items,    wherein said binding molecule is an antibody selected from the group    consisting of a monoclonal antibody, a polyclonal antibody, a    bi-specific antibody, an antibody-like molecule, a Fc-molecule, a    KIR-molecule, a ScFv and a Fab.-   15. The detection molecule according to any of the preceding items,    wherein said binding molecule is a peptide of 1-100 amino acid    residues without tertiary structure.-   16. The detection molecule according to any of the preceding items,    wherein said binding molecule is a MHC molecule or MHC complex.-   17. The detection molecule according to any of the preceding items,    wherein said binding molecule is a MHC class I complex.-   18. The detection molecule according to any of the preceding items,    wherein said binding molecule is a MHC class II complex.-   19. The detection molecule according to any of the preceding items,    wherein said binding molecule is a MHC-like molecule.-   20. The detection molecule according to any of the preceding items,    wherein said binding molecule is CD1, wherein said CD1 is selected    from the group consisting of CD1 CD1a, CD1b, CD1c, CD1d and CD1e.-   21. The detection molecule according to any of the preceding items,    wherein said binding molecule is a MHC Class I-like proteins;    including MIC A, MIC B, CD1d, HLA E, HLA F, HLA G, HLA H, ULBP-1,    ULBP-2, and ULBP-3.-   22. The detection molecule according to any of the preceding items,    wherein the target of the binding molecule is a cell-surface target.-   23. The detection molecule according to any of the preceding items,    wherein the target of the binding molecule is an intracellular    target.-   24. The detection molecule according to any of the preceding items,    wherein the target of the binding molecule is a receptor, such as a    cell-surface receptor, an intracellular receptor, a soluble receptor    or an extracellular receptor.-   25. The detection molecule according to any of the preceding items,    wherein the target of the binding molecule is a T-cell receptor.-   26. The detection molecule according to any of the preceding items,    wherein the target of the binding molecule is a B-cell receptor.-   27. The detection molecule according to any of the preceding items,    wherein the target of the binding molecule is CD4 of T cells.-   28. The detection molecule according to any of the preceding items,    wherein the target of the binding molecule is CD8 of T cells.-   29. The detection molecule according to any of the preceding items,    wherein the target of the binding molecule is CD20 of B cells.-   30. The detection molecule according to any of the preceding items,    wherein the target of the binding molecule is selected from the    group consisting of cancer cell markers, developmental markers, cell    cycle markers, proliferation markers, activation markers, hormones,    hormone receptors, intracellular markers, cluster of differentiation    (CD), cell surface markers, cytokines and cytokine receptors.-   31. The detection molecule according to any of the preceding items,    wherein the target of the binding molecule is selected from the    group consisting of CD2, CD3, CD4, CD5, CD8, CD9, CD27, CD28, CD30,    CD69, CD134 (OX40), CD137 (4-1BB), CD147, CDw150 (SLAM), CD152    (CTLA-4), CD153 (CD30L), CD40L (CD154), NKG2D, ICOS, HVEM, HLA Class    II, PD-1, Fas (CD95), FasL, CD40, CD48, CD58, CD70, CD72, B7.1    (CD80), B7.2 (CD86), B7RP-1, B7-H3, PD-L1, PD-L2, CD134L, CD137L,    ICOSL, LIGHT CD16, NKp30, NKp44, NKp46, NKp80, 2B4, KIR, LIR,    CD94/NKG2A, CD94/NKG2C, LFA-1, CD11a/18, CD54 (ICAM-1), CD106    (VCAM), CD49a,b,c,d,e,f/CD29 (VLA-4), CD11a, CD14, CD15, CD19, CD25,    CD30, CD37, CD49a, CD49e, CD56, CD27, CD28, CD45, CD45RA, CD45RO,    CD45RB, CCR7, CCRS, CD62L, CD75, CD94, CD99, CD107b, CD109, CD152,    CD153, CD154, CD160, CD161, CD178, CDw197, CDw217, Cd229, CD245,    CD247 and Foxp3.-   32. The detection molecule according to any of the preceding items,    wherein the target of the binding molecule is a cytokine selected    from the group consisting of TNFα, TNFβ, TNF, IFNα, IFNβ, IFNγ,    IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,    IL-10-20, IL-20-30, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36,    IL-37, IL-38, IL-39, IL-40, NFκB, chemokines including CC chemokines    (CCL1 to CCL-28), CXC chemokines (CXCL1 to CXCL17) C chemokines    (XCL-1 and -2) and CX3X chemokines (CX3CL1).-   33. The detection molecule according to any of the preceding items,    wherein the target of the binding molecule is a cancer cell marker    selected from the group consisting of HER2, CA125, Tyrosinase,    Melanoma-associated antigen (MAGE), abnormal products of Ras or p53,    Carcinoembryonic antigen, Muc-1, Epithelial tumor antigen, Carbonic    Anhydrase, VEGFR, EGFR, TRAIL and RAN KL.-   34. The detection molecule according to any of the preceding items,    wherein the target of the binding molecule is a stem cell marker    selected from the group consisting of Stro-1, CD146, CD105, CD44,    c-kit, Oct4, Sox-2, Klf4, EphB, Nestin and TWIST-1.-   35. The detection molecule according to any of the preceding items,    wherein the target of the binding molecule is a developmental marker    selected from the group consisting of Nanog, Oct4, Sox2, TEKT-1,    NANOS, c-kit, Sox9, Notch, Msx1, Msx2 and Col1.-   36. The detection molecule according to any of the preceding items,    wherein the target of the binding molecule is a proliferation marker    selected from the group consisting of CyclinA, CyclinB, PCNA, PC10,    p53, Mdm2, Cyclin D, Cyclin E, Rb, ARF and HDM2.-   37. The detection molecule according to any of the preceding items,    wherein the target of the binding molecule is an activation marker    selected from the group consisting of CD28, Tbet, Eomes, Blimp,    Bcl-6, CD27, MHC-II, TNF, IFN, Fizz1, ARG1 and CCL22R.-   38. The detection molecule according to any of the preceding items,    wherein the target of the binding molecule is an hormone selected    from the group consisting of estrogen, PTH, ADH, T3, ANP,    Epinephrine, Norepinephrine, Cortisol, Corticosterone, Aldosterone,    Progestin, EPO, Leptin, Insulin, Glucagon, T4, ACTH, FSH, oxytocin    and Calcitriol.-   39. The detection molecule according to any of the preceding items,    wherein the target of the binding molecule is an hormone receptor    selected from the group consisting of EstrogenR (ER), GLP-1R,    Thyroid receptor, Leptin receptor, Epinephrine receptor, Insulin    receptor and Glucagon receptor.-   40. The detection molecule according to any of the preceding items,    wherein the target of the binding molecule is a cluster of    differentiation (CD) molecule selected from the group consisting of    CD1-10, CD10-20, CD20-30, CD30-40, CD40-50, CD50-100, CD100-200,    CD200-300 and CD300-364.-   41. The detection molecule according to any of the preceding items,    wherein the target of the binding molecule is an intracellular    marker selected from the group consisting of Cyclins, Cytokines and    organelle markers (for example Apg12, Syntaxin, PAF-46, Histones,    Early endosome antigen, clathrin, tubulins, PAF49, FTCD).-   42. The detection molecule according to any of the preceding items,    wherein the target of the binding molecule is selected from the    group consisting of CD1, CD1a, CD1b, CD1c, CD1d, and MR1.-   43. The detection molecule according to any of the preceding items,    wherein the target of the binding molecule is selected from the    group consisting of targets included in Table 4 of the examples.-   44. The detection molecule according to any of the preceding items,    wherein said binding molecule is a surface-molecule receptor to a    cytokine receptor selected from the group consisting of interleukins    and TNF-like molecules.-   45. The detection molecule according to any of the preceding items,    wherein said label is any molecule, atom or signal the identity of    which can be determined.-   46. The detection molecule according to any of the preceding items,    wherein said label is unique to and/or specifies the binding    molecule or a group of binding molecules.-   47. The detection molecule according to any of the preceding items,    wherein said detection molecule comprises one label.-   48. The detection molecule according to any of the preceding items,    wherein said detection molecule comprises more than one label.-   49. The detection molecule according to any of the preceding items,    wherein said detection molecule comprises two or more labels, such    as 3 labels, 4 labels, 5 labels, 6 labels, 7 labels, 8 labels, 9    labels, 10 labels, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-25,    25-30, 30-40, 40-50, 50-75, 75-100 labels.-   50. The detection molecule according to any of the preceding items,    wherein said detection molecule comprises two or more labels and    each of said labels are identical to each other.-   51. The detection molecule according to any of the preceding items,    wherein said detection molecule comprises two or more labels and at    least two of said labels are different.-   52. The detection molecule according to any of the preceding items,    wherein said label is attached to the linker of the detection    molecule.-   53. The detection molecule according to any of the preceding items,    wherein said label is attached to the binding molecule of the    detection molecule.-   54. The detection molecule according to any of the preceding items,    wherein said label comprises a connector molecule (attachment    molecule) for attachment to the detection molecule.-   55. The detection molecule according to any of the preceding items,    wherein said label is a nucleic acid label.-   56. The detection molecule according to any of the preceding items,    wherein said label is a nucleic acid label selected from the group    consisting of a DNA label, an RNA label, and an artificial nucleic    acid label.-   57. The detection molecule according to any of the preceding items,    wherein said label is a nucleic acid label comprising one or more    nucleotides individually derived from one or more of DNA, RNA, and    an artificial nucleic acid.-   58. The detection molecule according to any of the preceding items,    wherein said artificial nucleic acid is selected from the group    consisting of XNA, LNA, PNA, GNA, TNA, HNA, CeNA, and    morpholino-nucleic acids.-   59. The detection molecule according to any of the preceding items,    wherein said label is a DNA label.-   60. The detection molecule according to any of the preceding items,    wherein said nucleic acid label comprises 1 to 1,000,000 nucleic    acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,    16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,    33, 34, 35, 36, 37, 38, 39, 40 nucleic acids; for example 1-3, 3-5,    5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-50, 50-60, 60-70,    70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150,    150-175, 175-200, 200-250, 250-300, 300-400, 400-500, 500-600,    600-700, 700-800, 800-900, 900-1000, 1000-1500, 1500-2000,    2000-3000, 3000-4000, 4000-5000, 5000-7500, 7500-10,000,    10,000-100,000, 100,000-1,000,000 nucleic acids.-   61. The detection molecule according to any of the preceding items,    wherein said label is a nucleic acid label comprising one or more of    -   a. barcode region,    -   b. 5′ first primer region (forward)    -   c. 3′ second primer region (reverse),    -   d. random nucleotide region,    -   e. connector molecule    -   f. stability-increasing components    -   g. short nucleotide linkers in between any of the        above-mentioned components    -   h. adaptors for sequencing    -   i. annealing region-   62. The detection molecule according to any of the preceding items,    wherein said label is a nucleic acid label comprising at least a    barcode region.-   63. The detection molecule according to any of the preceding items,    wherein said label is a nucleic acid label comprising at least a    barcode region, wherein said barcode region comprises a sequence of    consecutive nucleic acids.-   64. The detection molecule according to any of the preceding items,    wherein the barcode region of said nucleic acid comprises 1-3, 3-5,    5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-50, 50-60, 60-70,    70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150,    150-175, 175-200, 200-250, 250-300, 300-400, 400-500, 500-600,    600-700, 700-800, 800-900, 900-1000 nucleic acids.-   65. The detection molecule according to any of the preceding items,    wherein said barcode region comprises or consists of 5-10, 10-15,    15-20, 20-25, 25-30, 30-40, 40-45, 45-50 nucleic acids.-   66. The detection molecule according to any of the preceding items,    wherein said label is a nucleic acid label comprising at least a 3′    primer region, a barcode region, and a 5′ primer region.-   67. The detection molecule according to any of the preceding items,    wherein said label is a nucleic acid label comprising at least a 3′    primer region, a barcode region, and a 5′ primer region, wherein    said barcode region is designed to be amplified by e.g. PCR and    identified by e.g. sequencing.-   68. The detection molecule according to any of the preceding items,    wherein the primer regions of said nucleic acid label are identical    for subsets of detection molecules comprising different labels.-   69. The detection molecule according to any of the preceding items,    wherein said label is a nucleic acid label comprising a connector    molecule which is able to interact with a component on the linker    and/or binding molecule of the detection molecule.-   70. The detection molecule according to any of the preceding items,    wherein said nucleic acid label comprises a connector molecule which    is biotin.-   71. The detection molecule according to any of the preceding items,    wherein said nucleic acid label comprises a random nucleotide region    comprising 3-20 nucleotides, such as 3, 4, 5, 6, 7, 8, 9, 10, 11,    12, 13, 14, 15, 16, 17, 18, 19 or 20 nt.-   72. The detection molecule according to any of the preceding items,    wherein said nucleic acid label comprises one or more    stability-increasing components, such as HEG or TEG.-   73. The detection molecule according to any of the preceding items,    wherein a sample identifying sequence is attached to the nucleic    acid label such as by attachment to one of the primers capable of    binding to the primer regions of the nucleic acid label.-   74. The detection molecule according to any of the preceding items,    wherein said label is a peptide label.-   75. The detection molecule according to any of the preceding items,    wherein said peptide label comprises a stretch of consecutive amino    acid residues (coding region).-   76. The detection molecule according to any of the preceding items,    wherein said peptide label comprises a stretch of consecutive amino    acid residues (coding region) and a protease cleavage site.-   77. The detection molecule according to any of the preceding items,    wherein said peptide label comprises a stretch of consecutive amino    acid residues (coding region) and a protease cleavage site.-   78. The detection molecule according to any of the preceding items,    wherein said protease cleavage site in said peptide label is located    proximal to the linker that connects the label to the binding    molecule.-   79. The detection molecule according to any of the preceding items,    wherein said peptide label comprising a protease cleavage site    allows for cleavage of the stretch of consecutive amino acid    residues (coding region) and release thereof from the detection    molecule.-   80. The detection molecule according to any of the preceding items,    wherein said label is a peptide label comprising 2 or more    consecutive amino acids, such as 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9,    9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19,    19-20, 20-21, 21-22, 22-23, 23-24, 24-25, 25-26, 26-27, 27-28,    28-29, 29-30, 30-31, 31-32, 32-33, 33-34, 34-35, 35-36, 36-37,    37-38, 38-39, 39-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70,    70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 100-110, 110-120,    120-130, 130-140, 140-150, 150-160, 160-170,170-180, 180-190,    190-200, 200-225, 225-250, 250-275, 275-300, 300-350, 350-400,    400-450, 450-500, 500-600, 600-700, 700-800, 800-900, 900-1000,    1000-1500, 1500-2000, or more than 2000, consecutive amino acids.-   81. The detection molecule according to any of the preceding items,    wherein said peptide label comprises proteinogenic and/or    non-proteinogenic amino acids.-   82. The detection molecule according to any of the preceding items,    wherein said label is a fluorescent label (fluorophore label).-   83. The detection molecule according to any of the preceding items,    wherein said label is a fluorescent label selected from the group    consisting of fluorescein isothiocyanate (FITC), fluorescein (Flu)    derivates, rhodamine, tetramethylrhodamine, phycoerythrin,    R-phycoerythrin (RPE), allophycoerythrin, phycocyanin,    allophycocyanin, o-phthaldehyde, fluorescamine;    2-(4′-maleimidylanilino)naphthalene-6-sulfonic acid;    5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid;    Pyrene-1-butanoic acid; AlexaFluor 350 (7-amino-6-sulfonic    acid-4-methyl coumarin-3-acetic acid); AMCA (7-amino-4-methyl    coumarin-3-acetic acid); 7-hydroxy-4-methyl coumarin-3-acetic acid;    Marina Blue (6,8-difluoro-7-hydroxy-4-methyl coumarin-3-acetic    acid); 7-dimethylamino-coumarin-4-acetic acid; Fluorescamin-N-butyl    amine adduct; 7-hydroxy-coumarine-3-carboxylic acid; CascadeBlue    (pyrene-trisulphonic acid acetyl azide); Cascade Yellow; Pacific    Blue (6,8 difluoro-7-hydroxy coumarin-3-carboxylic acid);    7-diethylamino-coumarin-3-carboxylic acid;    N-(((4-azidobenzoyl)amino)ethyl)-4-amino-3,6-disulfo-1,8-naphthalimide;    Alexa Fluor 430; 3-perylenedodecanoic acid;    8-hydroxypyrene-1,3,6-trisulfonic acid;    12-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoic acid;    N,N′-dimethyl-N-(iodoacetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine;    Oregon Green 488 (difluoro carboxy fluorescein);    5-iodoacetamidofluorescein; propidium iodide-DNA adduct; Carboxy    fluorescein; 5- or 6-carboxyfluorescein; 6-(fluorescein)-5-(and    6)-carboxamido hexanoic acid; Texas Red, Princeton Red, Green    fluorescent protein (GFP) and analogues thereof; PerCP; AlexaFluor®    (AF), AF405, AF488,AF500, AF514, AF532, AF546, AF555, AF568, AF594,    AF610, AF633, AF635, AF647, AF680, AF700, AF710, AF750, AF800;    Quantum Dot based dyes, Qdot®525, Qdot®565, Qdot®585, Qdot®605,    Qdot®655, Qdot®705, Qdot®800; DyLight™ Dyes (Pierce) (DL); DL549,    DL649, DL680, DL800; Cy-Dyes, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7;    Fluorescent Proteins, RPE, PerCp, APC, Green fluorescent proteins;    GFP and GFP derivated mutant proteins; BFP, CFP, YFP, DsRed, T1,    Dimer2, mRFP1,MBanana, mOrange, dTomato, tdTomato, mTangerine,    mStrawberry, mCherry; Tandem dyes, RPE-Cy5, RPE-Cy5.5, RPE-Cy7,    RPE-AlexaFluor® tandem conjugates; RPE-Alexa610, RPE-TxRed,    APC-Aleca600, APC-Alexa610, APC-Alexa750, APC-Cy5 and APC-Cy5.5.-   84. The detection molecule according to any of the preceding items,    wherein said label is a phosphorescence label.-   85. The detection molecule according to any of the preceding items,    wherein said label is a bioluminescence label or chemoluminescence    label.-   86. The detection molecule according to any of the preceding items,    wherein said chemoluminescence label is selected from luminol,    isoluminol, acridinium esters, acridinium salt, theromatic    acridinium ester, 1,2-dioxetanes, oxalate ester, imidazole and    pyridopyridazines.-   87. The detection molecule according to any of the preceding items,    wherein said bioluminescence label is selected from the group    luciferin, luciferase and aequorin.-   88. The detection molecule according to any of the preceding items,    wherein said label is an enzymatic label.-   89. The detection molecule according to any of the preceding items,    wherein said label is an enzymatic label, wherein the enzyme    catalyze a reaction between chemicals in the near environment of the    labeling molecules, which results in one or more of producing a    light signal (chemi-luminescence) and precipitation of chromophor    dyes.-   90. The detection molecule according to any of the preceding items,    wherein said label is an enzymatic label selected from peroxidases,    malate dehydrogenase, staphylococcal nuclease, delta-5-steroid    isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate,    dehydrogenase, triose phosphate isomerase, horseradish peroxidase,    alkaline phosphatase, asparaginase, glucose oxidase,    beta-galactosidase, ribonuclease, urease, catalase,    glucose-6-phosphate dehydrogenase, glucoamylase and    acetylcholinesterase.-   91. The detection molecule according to any of the preceding items,    wherein said label is an enzymatic label selected from horse radish    peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase    (GAL), glucose-6-phosphate dehydrogenase,    beta-N-acetylglucosaminidase, β-glucuronidase, invertase, Xanthine    Oxidase, firefly luciferase and glucose oxidase (GO).-   92. The detection molecule according to any of the preceding items,    wherein said label is capable of reflection of light, such as gold,    plastic, glass, polystyrene and pollen.-   93. The detection molecule according to any of the preceding items,    wherein said label is capable of capable of absorption of light,    such as a chromophore or a dye.-   94. The detection molecule according to any of the preceding items,    wherein said label is capable of emission of light after excitation,    such as a fluorochrome.-   95. The detection molecule according to any of the preceding items,    wherein said label is a nanoparticle label.-   96. The detection molecule according to any of the preceding items,    wherein said label is an element.-   97. The detection molecule according to any of the preceding items,    wherein said label is selected from the group consisting of heavy    metal labels, isotope labels, radiolabels, radionuclide, stable    isotopes, chains of isotopes and single atoms.-   98. The detection molecule according to any of the preceding items,    wherein said label is a single atom selected from the group    consisting of zinc (Zn), iron (Fe), magnesium (Mg), any of the    lanthanides (Ln) including La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy,    Ho, Er, Tm, Yb and Lu; scandium (Sc) and yttrium (Y).-   99. The detection molecule according to any of the preceding items,    wherein said radioactivity labels comprises incorporated isotopes of    iodide, cobalt, selenium, tritium and/or phosphor.-   100. The detection molecule according to any of the preceding items,    wherein said label is a DNA fluorescing stain, such as Propidium    iodide, Hoechst stain, DAPI, DraQ5 or Acridine orange.-   101. The detection molecule according to any of the preceding items,    wherein said label comprises a nucleic acid label and at least a    second label.-   102. The detection molecule according to any of the preceding items,    wherein said label comprises a nucleic acid label and at least a    second label according to any of the preceding items.-   103. The detection molecule according to any of the preceding items,    wherein said label comprises a nucleic acid label and a fluorophore    label.-   104. The detection molecule according to any of the preceding items,    wherein said label comprises one type of label.-   105. The detection molecule according to any of the preceding items,    wherein said label comprises more than one type of label, such as    comprising 2 types of labels, for example comprising 3 types of    labels, such as comprising 4 types of labels, for example comprising    5 types of labels.-   106. The detection molecule according to any of the preceding items,    wherein said linker is a molecular entity and/or bond that connects    the binding molecule and the label.-   107. The detection molecule according to any of the preceding items,    wherein one or more of the binding molecules are covalently    associated with the one or more linkers.-   108. The detection molecule according to any of the preceding items,    wherein one or more of the binding molecules are non-covalently    associated with the one or more linkers.-   109. The detection molecule according to any of the preceding items,    wherein one or more labels are covalently associated with the one or    more linkers.-   110. The detection molecule according to any of the preceding items,    wherein one or more of labels are non-covalently associated with the    one or more linkers.-   111. The detection molecule according to any of the preceding items,    wherein the one or more labels and/or one or more binding molecules    are associated with a molecule in the one or more linkers, such as a    connector molecule, a sugar residue, a protein, an antibody, a DNA,    an aptamer, reactive groups, nucleophilic group, electrophilic    groups, radicals, or conjugated double bonds.-   112. The detection molecule according to any of the preceding items,    wherein the one or more linkers comprises one or more    multimerization domains.-   113. The detection molecule according to any of the preceding items,    wherein the one or more linkers comprises one or more scaffolds.-   114. The detection molecule according to any of the preceding items,    wherein the one or more linkers comprises one or more connectors.-   115. The detection molecule according to any of the preceding items,    wherein the one or more multimerization domains comprises at least    one scaffold and at least one connector.-   116. The detection molecule according to any of the preceding items,    wherein the binding molecule and/or the label is attached to the    linker via a streptavidin-biotin linkage.-   117. The detection molecule according to any of the preceding items,    wherein the one or more linkers comprise one or more optionally    substituted organic molecules.-   118. The detection molecule according to any of the preceding items,    wherein the optionally substituted organic molecule comprises one or    more functionalized cyclic structures.-   119. The detection molecule according to any of the preceding items,    wherein the one or more functionalized cyclic structures comprises    one or more benzene rings.-   120. The detection molecule according to any of the preceding items,    wherein the optionally substituted organic molecule comprises a    scaffold molecule comprising at least three reactive groups, or at    least three sites suitable for non-covalent attachment.-   121. The detection molecule according to any of the preceding items,    wherein the one or more linkers comprises one or more biological    cells and/or cell-like structures, such as antigen presenting cells    or dendritic cells.-   122. The detection molecule according to any of the preceding items,    wherein the one or more linkers comprises one or more membranes.-   123. The detection molecule according to any of the preceding items,    wherein the one or more membranes comprises liposomes or micelles.-   124. The detection molecule according to any of the preceding items,    wherein the one or more linkers comprises one or more polymers such    as one or more synthetic polymers.-   125. The detection molecule according to any of the preceding items,    wherein reactive groups involved in forming an association between    the multimerization domain and the binding molecule are located on    glutamate or aspartate residues, or on a vinyl sulfone activated    dextran.-   126. The detection molecule according to any of the preceding items,    wherein the one or more linker polymers are selected from the group    consisting of polysaccharides.-   127. The detection molecule according to any of the preceding items,    wherein the linker comprises one or more dextran moieties.-   128. The detection molecule according to any of the preceding items,    wherein the one or more dextran moieties are covalently attached to    one or more binding molecules.-   129. The detection molecule according to any of the preceding items,    wherein the one or more dextran moieties are non-covalently attached    to one or more binding molecules.-   130. The detection molecule according to any of the preceding items,    wherein the one or more dextran moieties are covalently attached to    one or more labels.-   131. The detection molecule according to any of the preceding items,    wherein the one or more dextran moieties are non-covalently attached    to one or more labels.-   132. The detection molecule according to any of the preceding items,    wherein reactive groups of the multimerization domains include    hydroxyls of polysaccharides such as dextrans-   133. The detection molecule according to any of the preceding items,    wherein the one or more linker dextran moieties are modified.-   134. The detection molecule according to any of the preceding items,    wherein the one or more linker dextran moieties are activated.-   135. The detection molecule according to any of the preceding items,    wherein the one or more linker dextran moieties are activated by    reaction of the dextran hydroxyls with divinyl sulfon.-   136. The detection molecule according to any of the preceding items,    wherein dextran is activated by a multistep reaction that results in    the decoration of the dextran with maleimide groups.-   137. The detection molecule according to any of the preceding items,    wherein the one or more linker dextran moieties comprises one or    more amino-dextrans.-   138. The detection molecule according to any of the preceding items,    wherein the one or more linker dextran moieties comprises one or    more amino-dextrans modified with divinyl sulfone.-   139. The detection molecule according to any of the preceding items,    wherein the one or more linker dextran moieties comprises one or    more dextrans with a molecular weight of from 1,000 to 50,000, such    as from 1,000 to 5,000, for example 5,000 to 10,000, such as from    10,000 to 15,000, for example 15,000 to 20,000, such as from 20,000    to 25,000, for example 25,000 to 30,000, such as from 30,000 to    35,000, for example 35,000 to 40,000, such as from 40,000 to 45,000,    for example 45,000 to 50,000.-   140. The detection molecule according to any of the preceding items,    wherein the one or more linker dextran moieties comprises one or    more dextrans with a molecular weight of from 50,000 to 150,000,    such as from 50,000 to 60,000, for example 60,000 to 70,000, such as    from 70,000 to 80,000, for example 80,000 to 90,000, such as from    90,000 to 100,000, for example 100,000 to 110,000, such as from    110,000 to 120,000, for example 120,000 to 130,000, such as from    130,000 to 140,000, for example 140,000 to 150,000.-   141. The detection molecule according to any of the preceding items,    wherein the one or more linker dextran moieties comprises one or    more dextrans with a molecular weight of from 150,000-270,000 such    as from 150,000 to 160,000, for example 160,000 to 170,000, such as    from 170,000 to 180,000, for example 180,000 to 190,000, such as    from 190,000 to 200,000, for example 200,000 to 210,000, such as    from 210,000 to 220,000, for example 220,000 to 230,000, such as    from 230,000 to 240,000, for example 240,000 to 250,000, such as    from 250,000 to 260,000, for example 260,000 to 270,000, such as    from 270,000 to 280,000, for example 280,000 to 290,000, such as    from 290,000 to 300,000, for example 300,000 to 310,000 such as from    310,000 to 320,000, for example 320,000 to 330,000 such as from    330,000 to 340,000, for example 340,000 to 350,000 such as from    350,000 to 360,000, for example 360,000 to 370,000 such as from    370,000 to 380,000, for example 380,000 to 390,000, such as from    390,000 to 400,000.-   142. The detection molecule according to any of the preceding items,    wherein the one or more linker dextran moieties are linear.-   143. The detection molecule according to any of the preceding items,    wherein the one or more linker dextran moieties are branched.-   144. The detection molecule according to any of the preceding items,    wherein the one or more linkers comprises a carboxy methyl dextran    and/or a dextran polyaldehyde and/or a carboxymethyl dextran lactone    and/or a cyclodextrin.-   145. The detection molecule according to any of the preceding items,    wherein the one or more linker synthetic polymers are selected from    the group consisting of PNA, polyimide and PEG.-   146. The detection molecule according to any of the preceding items,    wherein the one or more linkers comprises one or more entities    selected from the group consisting of an IgG domain, a coiled-coil    polypeptide structure, a DNA duplex, a nucleic acid duplex, PNA-PNA,    PNA-DNA, DNA-RNA.-   147. The detection molecule according to any of the preceding items,    wherein the one or more linkers, such as one or more multimerization    domains, comprises one or more avidins, such as one or more    streptavidins.-   148. The detection molecule according to any of the preceding items,    wherein the one or more streptavidins comprises one or more    tetrameric streptavidin variants.-   149. The detection molecule according to any of the preceding items,    wherein the one or more streptavidins comprises one or more    monomeric streptavidin variants.-   150. The detection molecule according to any of the preceding items,    wherein the one or more linkers comprises an antibody.-   151. The detection molecule according to any of the preceding items,    wherein the linker antibody is selected from the group consisting of    polyclonal antibody, monoclonal antibody, IgA, IgG, IgM, IgD, IgE,    IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, humanized antibody,    humanized monoclonal antibody, chimeric antibody, mouse antibody,    rat antibody, rabbit antibody, human antibody, camel antibody, sheep    antibody, engineered human antibody, epitope-focused antibody,    agonist antibody, antagonist antibody, neutralizing antibody,    naturally-occurring antibody, isolated antibody, monovalent    antibody, bispecific antibody, trispecific antibody, multispecific    antibody, heteroconjugate antibody, immunoconjugates,    immunoliposomes, labeled antibody, antibody fragment, domain    antibody, nanobody, minibody, maxibody, diabody, fusion antibody.-   152. The detection molecule according to any of the preceding items,    wherein the detection molecule comprises one or more organic    molecules selected from the group consisting of small organic    scaffold molecules, small organic molecules, steroids, peptides,    aromatic organic molecules, monocyclic structures, functionalized or    substituted benzene rings, dicyclic structures, polycyclic    structures, aliphatic molecules, monocyclic molecules, dicyclic    molecules, polycyclic molecules.-   153. The detection molecule according to any of the preceding items,    wherein the detection molecule comprises one or more monomeric    molecules able to polymerize; one or more biological polymers such    as one or more proteins; one or more small molecule scaffolds; one    or more supramolecular structure(s) such as one or more    nanoclusters; and/or one or more protein complexes.-   154. The detection molecule according to any of the preceding items,    wherein the linker of the detection molecule comprises one or more    beads.-   155. The detection molecule according to any of the preceding items,    wherein the linker is a bead coated with streptavidin, such as    streptavidin monomers or tetramers, and the one or more binding    molecules are biotinylated.-   156. The detection molecule according to any of the preceding items,    wherein the linker is a bead coated with polysaccharide, such as a    polysaccharide comprising dextran moieties.-   157. The detection molecule according to any of the preceding items,    wherein the one or more beads are selected from the groups    consisting of beads that carry electrophilic groups e.g. divinyl    sulfone activated polysaccharide, polystyrene beads that have been    functionalized with tosyl-activated esters, magnetic polystyrene    beads functionalized with tosyl-activated esters, and beads where    binding molecules have been covalently immobilized to these by    reaction of nucleophiles comprised within the binding molecules with    the electrophiles of the beads.-   158. The detection molecule according to any of the preceding items,    wherein the one or more beads is selected from the groups consisting    of sepharose beads, sephacryl beads, polystyrene beads, agarose    beads, polysaccharide beads, polycarbamate beads and any other kind    of beads that can be suspended in an aqueous buffer.-   159. The detection molecule according to any of the preceding items,    wherein the linker comprises one or more compounds selected from the    group consisting of agarose, sepharose, resin beads, glass beads,    pore-glass beads, glass particles coated with a hydrophobic polymer,    chitosan-coated beads, SH beads, latex beads, spherical latex beads,    allele-type beads, SPA bead, PEG-based resins, PEG-coated bead,    PEG-encapsulated bead, polystyrene beads, magnetic polystyrene    beads, glutathione agarose beads, magnetic bead, paramagnetic beads,    protein A and/or protein G sepharose beads, activated carboxylic    acid bead, macroscopic beads, microscopic beads, insoluble resin    beads, silica-based resins, cellulosic resins, cross-linked agarose    beads, polystyrene beads, cross-linked polyacrylamide resins, beads    with iron cores, metal beads, dynabeads, Polymethylmethacrylate    beads activated with NHS, streptavidin-agarose beads, polypropylene,    polyethylene, dextran, nylon, amylases, natural and modified    celluloses, nitrocellulose, polyacrylamides, gabbros, magnetite,    polymers, oligomers, non-repeating moieties, polyethylene glycol    (PEG), monomethoxy-PEG, mono-(C₁-C₁₀)alkoxy-PEG, aryloxy-PEG,    poly-(N-vinyl pyrrolidone)PEG, tresyl monomethoxy PEG, PEG    propionaldehyde, bis-succinimidyl carbonate PEG, polystyrene bead    crosslinked with divinylbenzene, propylene glycol homopolymers, a    polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated    polyols (e.g., glycerol), polyvinyl alcohol, dextran, aminodextran,    carbohydrate-based polymers, cross-linked dextran beads,    polysaccharide beads, polycarbamate beads, divinyl sulfone activated    polysaccharide, polystyrene beads that have been functionalized with    tosyl-activated esters, magnetic polystyrene beads functionalized    with tosyl-activated esters, streptavidin beads,    streptavidin-monomer coated beads, streptavdin-tetramer coated    beads, Streptavidin Coated Compel Magnetic beads, avidin coated    beads, dextramer coated beads, divinyl sulfone-activated dextran,    Carboxylate-modified bead, amine-modified beads, antibody coated    beads, cellulose beads, grafted co-poly beads, poly-acrylamide    beads, dimethylacrylamide beads optionally crosslinked with    N—N′-bis-acryloylethylenediamine, hollow fiber membranes,    fluorescent beads, collagen-agarose beads, gelatin beads,    collagen-gelatin beads, collagen-fibronectin-gelatin beads, collagen    beads, chitosan beads, collagen-chitosan beads, protein-based beads,    hydrogel beads, hemicellulose, alkyl cellulose, hydroxyalkyl    cellulose, carboxymethylcellulose, sulfoethylcellulose, starch,    xylan, amylopectine, chondroitin, hyarulonate, heparin, guar,    xanthan, mannan, galactomannan, chitin and chitosan.-   160. The detection molecule according to any of the preceding items,    wherein the multimerization domain comprises one or more beads    further comprises a linker moiety.-   161. The detection molecule according to any of the preceding items,    wherein the multimerization domain comprises one or more beads    comprising a linker moiety which is a flexible linker, a rigid    linker, a water-soluble linker or a cleavable linker.-   162. The detection molecule according to any of the preceding items,    wherein the one or more linkers comprises a dimerization domain.-   163. The detection molecule according to any of the preceding items,    wherein the one or more linkers comprises a trimerization domain.-   164. The detection molecule according to any of the preceding items,    wherein the one or more linkers comprises a tetramerization domain.-   165. The detection molecule according to any of the preceding items,    wherein the one or more linkers comprises a pentamerization domain.-   166. The detection molecule according to any of the preceding items,    wherein the pentamerization domain comprises a coiled-coil    polypeptide structure.-   167. The detection molecule according to any of the preceding items,    wherein the one or more linkers comprises a hexamerization domain,    such as a hexamerization domain comprises three IgG domains.-   168. The detection molecule according to any of the preceding items,    wherein the one or more linkers comprises a polyamide and/or a    polyethylene glycol and/or a polysaccharide and/or a sepharose.-   169. The detection molecule according to any of the preceding items,    wherein the one or more linkers have a molecular weight of less than    1,000 Da.-   170. The detection molecule according to any of the preceding items,    wherein the one or more linkers have a molecular weight of from    1,000 Da to preferably less than 10,000 Da.-   171. The detection molecule according to any of the preceding items,    wherein the one or more linkers have a molecular weight of from    10,000 Da to preferably less than 100,000 Da.-   172. The detection molecule according to any of the preceding items,    wherein the one or more linkers have a molecular weight of from    100,000 Da to preferably less than 1,000,000 Da.-   173. The detection molecule according to any of the preceding items,    wherein the one or more linkers have a molecular weight of more than    1,000,000 Da.-   174. The detection molecule according to any of the preceding items    further comprising one or more scaffolds, carriers, connectors    and/or linkers selected from the group consisting of streptavidin    (SA) and avidin and derivatives thereof, biotin, immunoglobulins,    antibodies (monoclonal, polyclonal, and recombinant), antibody    fragments and derivatives thereof, leucine zipper domain of AP-1    (jun and fos), hexa-his (metal chelate moiety), hexa-hat GST    (glutathione S-tranferase) glutathione affinity, Calmodulin-binding    peptide (CBP), Strep-tag, Cellulose Binding Domain, Maltose Binding    Protein, S-Peptide Tag, Chitin Binding Tag, Immuno-reactive    Epitopes, Epitope Tags, E2Tag, HA Epitope Tag, Myc Epitope, FLAG    Epitope, AU1 and AU5 Epitopes, Glu-Glu Epitope, KT3 Epitope, IRS    Epitope, Btag Epitope, Protein Kinase-C Epitope, VSV Epitope,    lectins that mediate binding to a diversity of compounds, including    carbohydrates, lipids and proteins, e.g. Con A (Canavalia    ensiformis) or WGA (wheat germ agglutinin) and tetranectin or    Protein A or G (antibody affinity).-   175. The detection molecule according to any of the preceding items,    wherein the binding molecule and/or the label comprises a connector    molecule, such as biotin, and the linker comprises a connector such    as streptavidin or another avidin connector.-   176. The detection molecule according to any of the preceding items,    wherein the binding molecule is linked to at least one of the one or    more multimerization domains by a non-covalent linker moiety, such    as natural dimerization or protein-protein interactions.-   177. The detection molecule according to any of the preceding items,    wherein the binding molecule is linked to at least one of the one or    more multimerization domains by a protein-protein interaction    selected from the group consisting of Fos/Jun interactions,    Acid/Base coiled coil structure based interactions, antibody/antigen    interactions, polynucleotide-polynucleotide interactions, synthetic    molecule-synthetic molecule interactions and protein-small molecule    interactions.-   178. The detection molecule according to any of the preceding items,    wherein the binding molecule is linked to at least one of the one or    more multimerization domains by natural dimerization selected from    the group consisting of antigen-antibody pairs, DNA-DNA    interactions, natural interactions, biotin and streptavidin.-   179. The detection molecule according to any of the preceding items,    wherein the detection molecule further comprises an enzyme capable    of catalysing the transfer of a cell surface moiety (e.g. a peptide    fragment or ‘peptide tag’) from a cell surface protein to the    binding molecule of the detection molecule, when said surface moiety    binds to the binding molecule.-   180. The detection molecule according to any of the preceding items,    comprising    -   a. a monomeric or a multimeric major histocompatibility complex        (MHC) molecule, such as a monomeric or multimeric peptide MHC        complex,    -   b. a linker comprising a multimerization domain and optionally        one or more connectors, and    -   c. a nucleic acid label.-   181. The detection molecule according to any of the preceding items,    comprising    -   a. a monomeric or a multimeric major histocompatibility complex        (MHC) molecule, such as a monomeric or multimeric peptide MHC        complex,    -   b. a linker comprising a multimerization domain and optionally        one or more connectors, and    -   c. a peptide label.-   182. The detection molecule according to any of the preceding items,    comprising    -   a. CD1, wherein said CD1 is selected from the group consisting        of CD1 CD1a, CD1b, CD1c, CD1d and CD1e,    -   b. a linker comprising a multimerization domain and optionally        one or more connectors, and    -   c. a nucleic acid label.-   183. The detection molecule according to any of the preceding items,    comprising    -   a. an anti-target molecule capable of associating with,        recognizing and/or binding to a predetermined marker molecule        (or target) on a cell type, wherein said marker molecule is        specific for a certain cell type    -   b. a linker (Li) comprising a multimerization domain and        optionally one or more connectors, and    -   c. a nucleic acid label.-   184. The detection molecule according to any of the preceding items,    wherein said linker comprising a multimerization domain and    optionally one or more connectors is a dextran optionally comprising    streptavidin or avidin, and said binding molecule and/or label    optionally comprises biotin.-   185. A kit of parts comprising    -   a. One or more detection molecules according to any of the        preceding items, and    -   b. one or more additional components.-   186. The kit of parts according to any of the preceding items,    wherein said one or more additional components comprise reagents for    detecting and/or amplifying the label of the detection molecule.-   187. The kit of parts according to any of the preceding items,    wherein said one or more additional components comprise reagents for    detecting and/or amplifying the nucleic acid label of the detection    molecule.-   188. The kit of parts according to any of the preceding items,    wherein said reagents for detecting the nucleic acid label of the    detection molecule comprises one or more primer sets capable of    amplifying the nucleic acid label.-   189. A detection method comprising the steps of    -   a. Combining a sample with at least one detection molecule;        wherein the detection molecule comprises a binding molecule        (BM), a linker (Li), and a label (La) according to any of the        preceding items; and wherein said sample comprises at least one        cell and/or entity,    -   b. Incubating the at least one detection molecule and the        sample;    -   c. Isolating and/or detecting the at least one detection        molecule of step b), and    -   d. Optionally determining the identity of the at least one        detection molecule of step c).-   190. The detection method according to any of the preceding items,    wherein in step b) the one or more detection molecules are allowed    to associate with, recognize, and/or bind to said at least one cell    and/or entity through their binding molecule.-   191. The detection method according to any of the preceding items,    wherein in step c) and d) said detection molecule is comprised in a    cell-detection molecule complex or an entity-detection molecule    complex.-   192. The detection method according to any of the preceding items,    wherein in step c) and d) said detection molecule is not comprised    in a cell-detection molecule complex or an entity-detection molecule    complex.-   193. The detection method according to any of the preceding items,    wherein in step c) and d) said detection molecule is no longer    comprised in a cell-detection molecule complex or an    entity-detection molecule complex, wherein said detection molecule    has previously interacted with a cell-detection molecule complex or    an entity-detection molecule complex.-   194. The detection method according to any of the preceding items,    wherein said cell-detection molecule complexes comprises a cell,    such as an immune cell, associated with or bound to a detection    molecule having a binding molecule specific for the immune cell.-   195. The detection method according to any of the preceding items,    wherein step c) comprises isolating and detecting the at least one    detection molecule.-   196. The detection method according to any of the preceding items,    wherein step c) comprises first isolating and then detecting the at    least one detection molecule.-   197. The detection method according to any of the preceding items,    wherein step c) comprises detecting the at least one detection    molecule.-   198. The detection method according to any of the preceding items,    wherein step c) comprises isolating the at least one detection    molecule.-   199. The detection method according to any of the preceding items,    wherein step c) comprises first detecting and then isolating the at    least one detection molecule.-   200. The detection method according to any of the preceding items,    wherein in step c) isolating comprises flow cytometry and/or FACS    sorting-   201. The detection method according to any of the preceding items,    wherein in step c) isolating comprises one or more steps of washing,    centrifugation and/or precipitation.-   202. The detection method according to any of the preceding items,    wherein in step c) isolating comprises one or more steps of    filtration.-   203. The detection method according to any of the preceding items,    wherein in step c) isolating comprises one or more steps of    application on an affinity column.-   204. The detection method according to any of the preceding items,    wherein in step c) isolating comprises sorting of cell populations    based on the functional response to a stimuli (responsive or    non-responsive population), such as cytokine secretion,    phosphorylation, calcium release.-   205. The detection method according to any of the preceding items,    wherein in step c) isolating comprises sorting of cell populations    based on phenotype, such as by linking a certain set of phenotypic    characteristics to the antigen-responsiveness.-   206. The detection method according to any of the preceding items,    wherein in step c) isolating comprises immobilization of the    detection molecule and/or cell-detection molecule complexes.-   207. The detection method according to any of the preceding items,    wherein said immobilization of the cell-detection molecule complexes    comprises precipitating cells, such as by centrifugation, by    immunoprecipitation, or any other means that precipitates the cells.-   208. The detection method according to any of the preceding items,    wherein said immobilization of the cell-detection molecule complexes    comprises binding the cell-detection molecule complexes to a bead, a    particle, another surface, an antibody or an MHC complex.-   209. The detection method according to any of the preceding items,    wherein said immobilization of the detection molecule and/or    cell-detection molecule complexes comprises hybridization onto an    array.-   210. The detection method according to any of the preceding items,    wherein said immobilization of the detection molecule and/or    cell-detection molecule complexes by hybridization onto an array    comprises a nucleic acid/nucleic acid-interaction between the    nucleic acid label of the detection molecule and an antisense    nucleic acid sequence in the array.-   211. The detection method according to any of the preceding items,    wherein said immobilization of the detection molecule and/or    cell-detection molecule complexes by hybridization onto an array    comprises a DNA/DNA-interaction between the DNA label of the    detection molecule and an antisense DNA in the array.-   212. The detection method according to any of the preceding items,    wherein said detecting in step c) and/or determining the identity in    step d) comprises one or more steps of adding primary antibodies    that bind to the immobilized detection molecule and/or    cell-detection molecule complexes and detecting said primary    antibodies directly wherein the primary antibody is labelled, or    indirectly by adding labelled secondary antibodies.-   213. The detection method according to any of the preceding items,    wherein said detecting in step c) and/or determining the identity in    step d) comprises one or more steps of detecting the immobilized    detection molecule and/or cell-detection molecule complexes by    monitoring read-out from a second label such as a fluorophore.-   214. The detection method according to any of the preceding items,    wherein said detecting in step c) and/or determining the identity in    step d) comprises one or more steps of determining the identity of    said label.-   215. The detection method according to any of the preceding items,    wherein said detecting in step c) and/or determining the identity in    step d) comprises interaction between ‘coating DNA’ on the cell    surface and the DNA label of the detection molecule.-   216. The detection method according to any of the preceding items,    wherein said detecting in step c) and/or determining the identity in    step d) comprises protease cleavage of the peptide label of the    detection molecule.-   217. The detection method according to any of the preceding items,    wherein said detecting in step c) and/or determining the identity in    step d) comprises transfer of a cell surface moiety to the detection    molecule (e.g. a ‘peptide tag’).-   218. The detection method according to any of the preceding items,    wherein said detecting in step c) and/or determining the identity in    step d) comprises detection of the label based on the physical    characteristics of the label, including mass, sequence, charge,    volume, size, dimensions, fluorescence, absorption, emission, NMR    spectra and others.-   219. The detection method according to any of the preceding items,    wherein said detecting in step c) and/or determining the identity in    step d) comprises amplification of the label.-   220. The detection method according to any of the preceding items,    wherein said detecting in step c) and/or determining the identity in    step d) comprises sequencing of the label (e.g. DNA sequencing,    peptide sequencing).-   221. The detection method according to any of the preceding items,    wherein said detecting in step c) and/or determining the identity in    step d) comprises amplification of the barcode sequence of a nucleic    acid label by PCR and/or sequencing of the barcode sequence.-   222. The detection method according to any of the preceding items,    wherein said sequencing comprises deep sequencing or next generation    sequencing.-   223. The detection method according to any of the preceding items,    wherein said detecting in step c) and/or determining the identity in    step d) comprises mass spectrometry.-   224. The detection method according to any of the preceding items,    wherein said detecting in step c) and/or determining the identity in    step d) comprises one or more of gel electrophoresis, gel    filtration, PAGE, column fractionation, PCR and QPCR.-   225. The detection method according to any of the preceding items,    said method further comprising one or more steps of providing a    sample, preferably a sample comprising at least one entity and/or at    least one cell.-   226. The detection method according to any of the preceding items,    said method further comprising one or more steps of pre-treatment of    the sample, and/or pre-treatment of cells of the sample.-   227. The detection method according to any of the preceding items,    said method further comprising one or more steps of separating    unbound detection molecules from cell- or entity-detection molecule    complexes.-   228. The detection method according to any of the preceding items,    said method further comprising one or more steps of removing unbound    detection molecules by washing and/or centrifuging.-   229. The detection method according to any of the preceding items,    said method further comprising one or more steps of single-cell    sorting and sequencing.-   230. The detection method according to any of the preceding items,    said method further comprising one or more steps of single-cell T    cell sorting of and single-cell TCR sequencing.-   231. The detection method according to any of the preceding items,    wherein said sample comprises one or more cells.-   232. The detection method according to any of the preceding items,    wherein said sample comprises at least one cell and/or entity to    which the binding molecule of the detection molecule is able to    associate with, recognize and/or bind.-   233. The detection method according to any of the preceding items,    wherein said sample is selected from the group consisting of a solid    sample, a fluid sample, a semifluid sample, a liquid sample, a    solubilised sample and a sample comprising dissociated cells of a    solid sample.-   234. The detection method according to any of the preceding items,    wherein said sample is selected from the group consisting of a    biofilm, a biopsy, a surgical sample, a tissue sample, a    microarray-fixed sample, a section such as a fresh section, a frozen    section and a FFPE section.-   235. The detection method according to any of the previous items,    wherein said sample is selected from the group consisting of blood,    whole blood, plasma, serum, Peripheral blood mononuclear cells    (PBMC), human PBMN (HPBMC), buffy coat, synovial fluid, bone marrow,    cerebrospinal fluid, saliva, lymph fluid, seminal fluid, urine,    stool, exudate, transdermal exudates, pharyngeal exudates, nasal    secretions, sputum, sweat, bronchoalveolar lavage, tracheal    aspirations, fluid from joints, vitreous fluid, vaginal or urethral    secretions or semen.-   236. The detection method according to any of the previous items,    wherein said sample comprises cell populations isolated from a    fluid, a semifluid sample or a solid sample.-   237. The detection method according to any of the previous items,    wherein said cell is selected from the group consisting of immune    cells, lymphocytes, monocytes, dendritic cells, T-cells, B-cells and    NK cells-   238. The detection method according to any of the previous items,    wherein said cell is a T-cell, such as a T cell selected from the    group consisting of CD4+ T cells, CD8+ T cells, αβ T cells and    invariant γδ T cells.-   239. The detection method according to any of the previous items,    wherein said cell is an antigen-specific T-cell or    antigen-responsive T cell.-   240. The detection method according to any of the previous items,    wherein said cell comprises T-cell receptors.-   241. The detection method according to any of the previous items,    wherein said cell is a cancer cell.-   242. The detection method according to any of the previous items,    wherein said sample is derived from an organ selected from the group    consisting of lymph nodes, kidney, liver, skin, brain, heart,    muscles, bone marrow, skin, skeleton, lungs, the respiratory tract,    spleen, thymus, pancreas, exocrine glands, bladder, endocrine    glands, reproduction organs including the phallopian tubes, eye,    ear, vascular system, the gastroinstestinal tract including small    intestines, colon, rectum, canalis analis and prostate gland.-   243. The detection method according to any of the previous items,    wherein the surface of sample cells is coated with proteases capable    of cleaving a peptide label, for example by adding antibody-protease    conjugates where the antibody recognizes a particular cell surface    structure.-   244. The detection method according to any of the previous items,    wherein the surface of sample cells is coated with DNA    oligonucletides (“coating DNA”), for example by adding antibody-DNA    conjugates where the antibody recognizes a particular cell surface    structure.-   245. A method for detecting antigen-specific T cells in a sample,    said method comprising providing a detection molecule and a    detection method according to any of the preceding items.-   246. A method for detecting specific cells in a sample, said method    comprising providing a detection molecule and a detection method    according to any of the preceding items.-   247. A method for diagnosing a disease, said method comprising    providing a detection molecule and a detection method according to    any of the preceding items.-   248. A method for diagnosing a disease according to the previous    items, wherein said disease is selected from the group consisting of    cancer, Cancerous diseases, infectious diseases, Infectious diseases    caused by virus, Infectious diseases caused by bacteria, Infectious    diseases caused by fungus, Parasitic diseases, Allergic diseases,    Transplantation-related diseases and Autoimmune and inflammatory    diseases.-   249. A method for detecting the presence and/or abundance of a    certain cell or cell type in a sample, said method comprising    providing a detection molecule and a detection method according to    any of the preceding items, wherein said detection molecule    comprises a binding molecule capable of associating specifically    with the cell or cell type in said sample-   250. A method for investigating the binding characteristics of a    certain cell or cell type in a sample, said method comprising    providing a detection molecule and a detection method according to    any of the preceding items, wherein said detection molecule    comprises a binding molecule capable of associating specifically    with a known target.-   251. A method for the identification of epitopes comprising    providing a detection molecule and a detection method according to    any of the preceding items-   252. A method for vaccine development comprising providing a    detection molecule and a detection method according to any of the    preceding items.-   253. A method for measuring immune reactivity after vaccination    comprising providing a detection molecule and a detection method    according to any of the preceding items.-   254. A method for development of immune-therapeutics comprising    providing a detection molecule and a detection method according to    any of the preceding items.

The invention claimed is:
 1. A composition for analysis of a cellpopulation, the composition comprising from 5 to 1,000,000 sets ofdetection molecules, wherein each of the detection molecules of a setcomprise a. at least two identical binding molecules, b. amultimerization domain coupled with each of the at least two identicalbinding molecules, wherein each binding molecule is coupled to themultimerization domain via a separate connector molecule, and whereinsaid multimerization domain is selected from the group consisting ofpolysaccharides, dextran moieties, avidins, streptavidins, andstreptactin, c. a first label, where the first label comprises at leastone nucleic acid label coupled with the multimerization domain or withat least one of said at least two identical binding molecules, whereinsaid nucleic acid label is 30-200 nucleotides in length and comprises i)a 5′ first primer region (forward), a barcode region and a 3′ secondprimer region (reverse), and ii) a random nucleotide region, whereinsaid barcode region comprises a sequence that is common within the setbut uniquely represents the set within the composition, wherein saidrandom nucleotide region comprises a sequence that is unique within thecomposition, and d. a second label, wherein the second label is selectedfrom the group consisting of fluorophores, chromophores, and peptides,and wherein the second label is coupled with the multimerization domainor with at least one of said at least two identical binding molecules,wherein said binding molecules of each of said sets recognize and/orbind to a T cell receptor or a B cell receptor specific for a given cellor cell type of said cell population.
 2. The composition according toclaim 1, wherein said nucleic acid label is selected from the groupconsisting of a DNA label, an RNA label, and an artificial nucleic acidlabel.
 3. The composition according to claim 1, wherein said nucleicacid label further comprises one or more of a. a connector molecule b.one or more stability-increasing components c. short nucleotide linkersin between any of the above-mentioned components d. adaptors forsequencing, and e. an annealing region.
 4. The composition according toclaim 1, wherein said binding molecules are selected from the groupconsisting of an antibody, an antibody mimetic, an antibody-likemolecule, an MHC complex, an MHC-like molecule, CD1, a peptide, apeptide aptamer, a nucleic acid aptamer, a DNA aptamer, an RNA aptamer,and a lymphocyte receptor ligand.
 5. The composition according to claim1, wherein said multimerization domain comprises a polysaccharide, andwherein said connector molecules comprise streptavidins and/or avidins.6. The composition according to claim 5, wherein said multimerizationdomain comprises dextran.
 7. The composition according to claim 1,wherein said binding molecules and/or first or second label comprisesbiotin.
 8. The composition according to claim 1, wherein the at leasttwo binding molecules of at least one of said sets comprise CD1, whereinsaid CD1 is selected from the group consisting of CD1a, CD1b, CD1c, CD1dand CD1e, and wherein said barcode region serves as an identificationtag for said CD1 within the composition.
 9. The composition according toclaim 1, wherein the at least two binding molecules of at least one ofsaid sets comprise a major histocompatibility complex (MHC) molecule, orone or more MHC-like molecules, and wherein said barcode region of theset serves as an identification tag for said MHC molecule or saidMHC-like molecule within the composition.
 10. The composition accordingto claim 9, wherein the at least two binding molecules comprise two ormore MHC molecules linked by said multimerization domain; wherein saidfirst label and/or second label are linked to said multimerizationdomain, or to one or more of said MHC molecules, wherein saidmultimerization domain is a dextran multimerization domain, and whereinsaid connector molecules comprise one or more streptavidins and/oravidins.
 11. The composition according to claim 1, wherein saidmultimerization domain is a dextran multimerization domain with saidconnector molecules comprising one or more streptavidins and/or avidins;and wherein said at least two binding molecules and said first labeland/or second label are attached to said dextran multimerization domain.12. The composition according to claim 1, wherein said multimerizationdomain comprises streptavidin and said binding moieties, first label,and/or second label comprise biotin.
 13. The composition according toclaim 1, wherein the barcode region of said nucleic acid label consistsof 3 to 30 nucleotides.
 14. The composition according to claim 1,wherein said multimerization domain is selected from the groupconsisting of polysaccharides, dextran moieties, avidins, andstreptavidins.
 15. The composition according to claim 1, wherein saidnucleic acid label is a DNA label.
 16. The composition according toclaim 1, wherein each nucleic acid label is sufficiently distinct fromother nucleic acid labels in said composition to allow specificidentification of a given nucleic acid barcode, such that saidindividual nucleic acid labels are distinguishable from each other. 17.The composition according to claim 1, wherein said at least two bindingmolecules comprise two or more MHC molecules, wherein the two or moreMHC molecules are linked by said multimerization domain, and whereinsaid multimerization domain comprises one or more streptavidins and/oravidins.
 18. The composition according to claim 1, wherein the bindingmolecules of at least one of said sets are selected from the groupconsisting of MHC molecules, MHC-peptide complexes, MHC-like molecules,CD1a, CD1b, CD1c, MR1, and HLA-E.
 19. The composition according to claim4, wherein the binding molecules are selected from the group consistingof peptides, proteins, antigens, MHC molecules, and MHC-like molecules.20. The composition according to claim 1, wherein said nucleic acidlabel further comprises an annealing region.
 21. The compositionaccording to claim 1, wherein said binding molecules of each of saidsets associate with, are recognized by, and bind to T cell receptors ofspecific T cells or T cell populations.
 22. The composition according toclaim 1, wherein said binding molecules of each of said sets associatewith, are recognized by, and bind to B cell receptors of specific Bcells or B cell populations.
 23. The composition according to claim 1,wherein said second label comprises one or more fluorophores.
 24. Thecomposition according to claim 23, wherein the one or more fluorophoresare suitable for use in fluorescence activated cell sorting.
 25. Thecomposition according to claim 1, wherein said random nucleotide regionis positioned between said 5′ first primer region and said 3′ secondprimer region.
 26. The composition according to claim 21, wherein saidbinding molecules of each of said sets bind to differentantigen-specific T-cells or antigen-specific T cell populations.
 27. Thecomposition according to claim 21, wherein said binding molecules ofeach of said sets bind to different antigen-specific B-cells orantigen-specific B cell populations.